Digital Assay

ABSTRACT

The invention relates to methods and compositions for the detection of targets in a sample.

PRIORITY DATA

This application claims the benefit of priority of U.S. ProvisionalApplication Nos. 60/332,519, filed Nov. 21, 2001, and 60/384,731, filedMay 31, 2002. Application Nos. 60/332,519 and 60/384,731 areincorporated by reference herein in their entirety for any purpose.

FIELD OF THE INVENTION

The invention relates to methods and compositions for the detection oftargets in a sample.

BACKGROUND

The detection of the presence or absence of one or more target sequencesin a sample containing one or more target sequences is commonlypracticed. For example, the detection of cancer and many infectiousdiseases, such as AIDS and hepatitis, routinely includes screeningbiological samples for the presence or absence of diagnostic nucleicacid sequences. Also, detecting the presence or absence of nucleic acidsequences is often used in forensic science, paternity testing, geneticcounseling, and organ transplantation.

SUMMARY OF THE INVENTION

In certain embodiments, methods for quantitating a target are provided.In certain embodiments, the methods comprise forming a reaction mixturecomprising: a sample possibly containing the target; a codeable label;one or more target-specific probes, wherein each target-specific probebinds specifically to the target under selective binding conditions; anda separating moiety. In certain embodiments, the methods furthercomprise treating the reaction mixture under reaction conditions suchthat a detectable complex is produced when the target is present, andsuch that a detectable complex is not produced when the target isabsent, and wherein the detectable complex comprises the codeable label,the target-specific probe, and the separating moiety. In certainembodiments, the methods further comprise separating the detectablecomplex from codeable labels that are not included in the detectablecomplex, and quantitating the target by counting the number of codeablelabels.

In certain embodiments, methods for quantitating at least two differentparticular targets are provided. In certain embodiments, the methodscomprise forming a reaction mixture comprising: a sample possiblycontaining two or more different particular targets; a differentcodeable label specific for each different particular target; one ormore different target-specific probes specific for each differentparticular target that bind specifically to the target under selectivebinding conditions; and a separating moiety. In certain embodiments, themethods further comprise treating the reaction mixture under reactionconditions such that when a particular target is present, a detectablecomplex is produced, which comprises the codeable label specific for theparticular target, the target-specific probe specific for the particulartarget, and the separating moiety, and when a particular target isabsent, a detectable complex is not produced. In certain embodiments,the methods further comprise separating any detectable complexesproduced from codeable labels that are not included in the detectablecomplex, and quantitating each of the different particular targets bycounting the number of codeable labels specific for each of thedifferent particular targets.

In certain embodiments, methods for quantitating at least two differenttarget nucleic acid sequences in a sample are provided. In certainembodiments, the methods comprise forming a ligation reaction mixture bycombining the sample with a different probe set specific for each of theat least two different target nucleic acid sequences. In certainembodiments, each probe set comprises (a) at least one separating bead,comprising a magnetic particle and a first target-specific probe, and(b) at least one detecting bead, comprising a codeable label, and asecond target-specific probe; wherein the target-specific probes in eachset are suitable for ligation together when hybridized adjacent to oneanother on a complementary target sequence. In certain embodiments, themethods further comprise subjecting the ligation reaction mixture to aligation reaction, wherein adjacently hybridizing complementarytarget-specific probes are ligated to one another to form a ligationproduct comprising the separating bead and the detecting bead. Incertain embodiments, the methods further comprise separating anyligation product from unligated separating and detecting beads. Incertain embodiments, the methods further comprise quantitating each ofthe at least two different target nucleic acid sequences by counting thenumber of codeable labels.

In certain embodiments, kits for detecting target nucleic acid sequencesin a sample are provided. In certain embodiments, the kits comprise adifferent bead set specific for each of the target nucleic acidsequences. In certain embodiments, each different bead set comprises (a)at least one separating bead, comprising a magnetic particle, a firstcodeable label comprising two or more labels, and a firsttarget-specific probe, wherein the first codeable label is specific forthe first target-specific probe, and (b) at least one detecting bead,comprising a second codeable label comprising a set of two or morelabels, and a second target-specific probe, wherein the second codeablelabel is specific for the second target-specific probe; and wherein thefirst codeable label is detectably different from the second codeablelabel. In certain embodiments, the target-specific probes in each setare suitable for ligation together when hybridized adjacent to oneanother on a complementary target sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a probe set according to certain embodiments of theinvention.

FIG. 2 illustrates methods for differentiating between two potentialalleles in a target locus using certain embodiments of the invention.

FIG. 2(A) shows: (i) two different probe sets that have different firsttarget-specific probes, A and B, that differ in their pivotal complement(T on the A probe and C on the B probe), and that have the same secondtarget-specific probe, Z, and (ii) a target sequence, comprising pivotalnucleotide A.

FIG. 2(B) shows the three target-specific probes annealed to the target.The sequence-specific portion of probe A is fully complementary with the3′ target region including the pivotal nucleotide. The pivotalcomplement of probe B is not complementary with the 3′ target region.The sequence-specific portion of probe B, therefore, contains abase-pair mismatch at the 3′ end. The sequence-specific portion of probeZ is fully complementary to the 5′ target region.

FIG. 2(C) shows ligation of target-specific probes A and Z to formligation product A-Z. Probes B and Z are not ligated together to form aligation product due to the mismatched pivotal complement on probe B.

FIG. 2( d) shows denaturing the double-stranded molecules to release theA-Z ligation product and unligated probes B and Z.

FIG. 3 illustrates certain potential binary and ternary codes using twocolors of labels according to certain embodiments.

FIG. 4 illustrates certain combinations of sets of labels (codes) whenone uses a two color binary code with a probe set according to certainembodiments. FIG. 4 also depicts the number of potential probe set codesaccording to certain embodiments when two ternary colors, 10 binarycolors, or 6 ternary colors are used.

FIG. 5 depicts exemplary alternative splicing.

FIG. 6 depicts certain embodiments for detecting splice variants.

FIG. 7 illustrates certain exemplary embodiments in which a first targetspecific probe and a second target specific probe are ligated afterhybridizing to a target molecule in a sample.

FIG. 8 illustrates certain exemplary embodiments in which separatingmoieties are separated from codeable labels and detectable complexes.

FIG. 9 illustrates certain embodiments of detecting of detectablecomplexes that have been separated from the sample.

FIG. 10 illustrates certain exemplary embodiments in which separatingmoieties are separated from codeable labels and detectable complexes.

FIG. 11 illustrates certain exemplary embodiments in which ligateddetectable complexes are separated from unligated codeable labels.

FIG. 12 illustrates certain exemplary embodiments in which ligateddetectable complexes are detected within the same vessel as the sampleand ligation reaction.

FIG. 13 illustrates certain exemplary embodiments in which a groove isincluded on the inner surface of the detection vessel for assisting inaligning ligated detectable complexes for detection.

FIG. 14( a) illustrates a probe set according to certain embodiments ofthe invention.

FIG. 14( b) illustrates two probe sets, ligation of the probe sets, anddetection of probe sets according to certain embodiments of theinvention.

FIG. 14 (c) illustrates a probe set according to certain embodiments ofthe invention.

FIG. 15 depicts results from a Taqman™ analysis of ligated detectablecomplexes comprising beads and ligation products that do not comprisebeads.

FIG. 16 shows photographs of detectable complexes obtained afterligation reactions.

FIG. 17 depicts the results of Taqman analyses of ligated detectablecomplexes produced in different concentrations of target molecules.

FIG. 18 illustrates certain exemplary embodiments of separation ofunpaired nonmagnetic beads from magnetic beads and detectable complexesby continuous flow, and a subsequent counting of detectable complexes byflow cytometry.

FIG. 19 illustrates certain exemplary embodiments of separation ofunpaired nonmagnetic beads from magnetic beads and detectable complexesby continuous flow, removal of unpaired magnetic beads by the differencein drag between detectable complexes and unpaired magnetic beads, andsubsequent counting of detectable complexes by flow cytometry.

FIG. 20 illustrates certain exemplary embodiments of separation ofunpaired nonmagnetic beads from magnetic beads and detectable complexesby continuous flow, separation of unpaired magnetic beads by sizefiltration, and subsequent counting of detectable complexes by flowcytometry.

FIG. 21 illustrates certain exemplary embodiments of a separation methodemploying magnetic beads and biotin-coated beads.

FIG. 22 illustrates certain exemplary embodiments of a ligation reactionemploying probes comprising addressable portions.

FIG. 23 illustrates certain exemplary embodiments in which a ligationproduct is attached to beads using hairpin structures.

FIG. 24 illustrates certain exemplary embodiments in which a ligationproduct is attached to beads using linking oligonucleotides.

FIG. 25 illustrates certain exemplary embodiments of an oligonucleotideligation (OLA) assay with a biotin molecule.

FIG. 26 illustrates certain exemplary embodiments of a codeable labelattached to an oligonucleotide that hybridizes to a ligation product.

FIG. 27 illustrates certain exemplary embodiments of separatingdetectable complexes from codeable labels not in detectable complexes.

FIG. 28 illustrates certain exemplary embodiments of separatingdetectable complexes from codeable labels not in detectable complexesusing a tube within a tube.

FIG. 29 illustrates certain exemplary embodiments of a codeable labelattached to a hairpin structure that hybridizes and ligates to aligation product.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included,”is not limiting. Also, the use of the term “portion” may include part ofa moiety or the entire moiety.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

DEFINITIONS AND TERMS

The term “nucleotide base”, as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, naturally occurring nucleotide bases adenine,guanine, cytosine, uracil, thymine, and analogs of the naturallyoccurring nucleotide bases, e.g., 7-deazaadenine, 7-deazaguanine,7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6-Δ2-isopentenyladenine(6iA), N6-Δ2-isopentenyl-2-methylthioadenine (2 ms6iA),N2-dimethylguanine (dmG), 7-methylguanine (7mG), inosine, nebularine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see, e.g., U.S. Pat. Nos.6,143,877 and 6,127,121 and PCT published application WO 01/38584),ethenoadenine, indoles such as nitroindole and 4-methylindole, andpyrroles such as nitropyrrole. Certain exemplary nucleotide bases can befound, e.g., in Fasman, 1989, Practical Handbook of Biochemistry andMolecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., and thereferences cited therein.

The term “nucleotide”, as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —R₂ or halogen groups, where each R isindependently H, C₁-C₆ alkyl or C₅-C₁₄ aryl. Exemplary riboses include,but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose, 2′-deoxy-3′-αminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352; andWO 99/14226). Exemplary LNA sugar analogs within a polynucleotideinclude, but are not limited to, the structures:

where B is any nucleotide base.

Modifications at the 2′- or 3′-position of ribose include, but are notlimited to, hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy,butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino,alkylamino, fluoro, chloro and bromo. Nucleotides include, but are notlimited to, the natural D optical isomer, as well as the L opticalisomer forms (see, e.g., Garbesi (1993) Nucl. Acids Res. 21:4159-65;Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata, (1993) NucleicAcids Symposium Ser. No. 29:69-70). When the nucleotide base is purine,e.g. A or G, the ribose sugar is attached to the N⁹-position of thenucleotide base. When the nucleotide base is pyrimidine, e.g. C, T or U,the pentose sugar is attached to the N¹-position of the nucleotide base,except for pseudouridines, in which the pentose sugar is attached to theC5 position of the uracil nucleotide base (see, e.g., Kornberg andBaker, (1992) DNA Replication, 2^(nd) Ed., Freeman, San Francisco,Calif.).

One or more of the pentose carbons of a nucleotide may be substitutedwith a phosphate ester having the formula:

where α is an integer from 0 to 4. In certain embodiments, α is 2 andthe phosphate ester is attached to the 3′- or 5′-carbon of the pentose.In certain embodiments, the nucleotides are those in which thenucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analogthereof. “Nucleotide 5′-triphosphate” refers to a nucleotide with atriphosphate ester group at the 5′ position, and are sometimes denotedas “NTP”, or “dNTP” and “ddNTP” to particularly point out the structuralfeatures of the ribose sugar. The triphosphate ester group may includesulfur substitutions for the various oxygens, e.g. α-thio-nucleotide5′-triphosphates. For a review of nucleotide chemistry, see: Shabarova,Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH,New York, 1994.

The term “nucleotide analog”, as used herein, refers to embodiments inwhich the pentose sugar and/or the nucleotide base and/or one or more ofthe phosphate esters of a nucleotide may be replaced with its respectiveanalog. In certain embodiments, exemplary pentose sugar analogs arethose described above. In certain embodiments, the nucleotide analogshave a nucleotide base analog as described above. In certainembodiments, exemplary phosphate ester analogs include, but are notlimited to, alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, etc., and mayinclude associated counterions.

Also included within the definition of “nucleotide analog” arenucleotide analog monomers which can be polymerized into polynucleotideanalogs in which the DNA/RNA phosphate ester and/or sugar phosphateester backbone is replaced with a different type of internucleotidelinkage. Exemplary polynucleotide analogs include, but are not limitedto, peptide nucleic acids, in which the sugar phosphate backbone of thepolynucleotide is replaced by a peptide backbone.

As used herein, the terms “polynucleotide”, “oligonucleotide”, and“nucleic acid” are used interchangeably and mean single-stranded anddouble-stranded polymers of nucleotide monomers, including2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked byinternucleotide phosphodiester bond linkages, or internucleotideanalogs, and associated counter ions, e.g., H⁺, NH₄ ⁺, trialkylammonium,Mg²⁺, Na⁺ and the like. A nucleic acid may be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof. The nucleotide monomer units may comprise any of thenucleotides described herein, including, but not limited to, naturallyoccurring nucleotides and nucleotide analogs. Nucleic acids typicallyrange in size from a few monomeric units, e.g. 5-40 when they aresometimes referred to in the art as oligonucleotides, to severalthousands of monomeric nucleotide units. Unless denoted otherwise,whenever a nucleic acid sequence is represented, it will be understoodthat the nucleotides are in 5′ to 3′ order from left to right and that“A” denotes deoxyadenosine or an analog thereof, “C” denotesdeoxycytidine or an analog thereof, “G” denotes deoxyguanosine or ananalog thereof, and “T” denotes thymidine or an analog thereof, unlessotherwise noted.

Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA,mRNA, rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained fromsubcellular organelles such as mitochondria or chloroplasts, and nucleicacid obtained from microorganisms or DNA or RNA viruses that may bepresent on or in a biological sample.

Nucleic acids may be composed of a single type of sugar moiety, e.g., asin the case of RNA and DNA, or mixtures of different sugar moieties,e.g., as in the case of RNA/DNA chimeras. In certain embodiments,nucleic acids are ribopolynucleotides and 2′-deoxyribopolynucleotidesaccording to the structural formulae below:

wherein each B is independently the base moiety of a nucleotide, e.g., apurine, a 7-deazapurine, a pyrimidine, or an analog nucleotide; each mdefines the length of the respective nucleic acid and can range fromzero to thousands, tens of thousands, or even more; each R isindependently selected from the group comprising hydrogen, halogen, —R″,—OR″, and —NR″R″, where each R″ is independently (C1-C6) alkyl or(C5-C14) aryl, or two adjacent Rs are taken together to form a bond suchthat the ribose sugar is 2′,3′-didehydroribose; and each R′ isindependently hydroxyl or

where α is zero, one or two.

In certain embodiments of the ribopolynucleotides and2′-deoxyribopolynucleotides illustrated above, the nucleotide bases Bare covalently attached to the C1′ carbon of the sugar moiety aspreviously described.

The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” mayalso include nucleic acid analogs, polynucleotide analogs, andoligonucleotide analogs. The terms “nucleic acid analog”,“polynucleotide analog” and “oligonucleotide analog” are usedinterchangeably and, as used herein, refer to a nucleic acid thatcontains at least one nucleotide analog and/or at least one phosphateester analog and/or at least one pentose sugar analog. Also includedwithin the definition of nucleic acid analogs are nucleic acids in whichthe phosphate ester and/or sugar phosphate ester linkages are replacedwith other types of linkages, such as N-(2-aminoethyl)-glycine amidesand other amides (see, e.g., Nielsen et al., 1991, Science 254:1497-1500; WO 92/20702; U.S. Pat. No. 5,719,262; U.S. Pat. No.5,698,685;); morpholinos (see, e.g., U.S. Pat. No. 5,698,685; U.S. Pat.No. 5,378,841; U.S. Pat. No. 5,185,144); carbamates (see, e.g., Stirchak& Summerton, 1987, J. Org. Chem. 52: 4202); methylene(methylimino) (see,e.g., Vasseur et al., 1992, J. Am. Chem. Soc. 114: 4006);3′-thioformacetals (see, e.g., Jones et al., 1993, J. Org. Chem. 58:2983); sulfamates (see, e.g., U.S. Pat. No. 5,470,967);2-aminoethylglycine, commonly referred to as PNA (see, e.g., Buchardt,WO 92/20702; Nielsen (1991) Science 254:1497-1500); and others (see,e.g., U.S. Pat. No. 5,817,781; Frier & Altman, 1997, Nucl. Acids Res.25:4429 and the references cited therein). Phosphate ester analogsinclude, but are not limited to, (i) C₁-C₄ alkylphosphonate, e.g.methylphosphonate; (ii) phosphoramidate; (iii) C₁-C₆alkyl-phosphotriester; (iv) phosphorothioate; and (v)phosphorodithioate.

The terms “annealing” and “hybridization” are used interchangeably andmean the base-pairing interaction of one nucleic acid with anothernucleic acid that results in formation of a duplex, triplex, or otherhigher-ordered structure. In certain embodiments, the primaryinteraction is base specific, e.g., A/T and G/C, by Watson/Crick andHoogsteen-type hydrogen bonding. In certain embodiments, base-stackingand hydrophobic interactions may also contribute to duplex stability.

The term “variant” as used herein refers to any alteration of a protein,including, but not limited to, changes in amino acid sequence,substitutions of one or more amino acids, addition of one or more aminoacids, deletion of one or more amino acids, and alterations to the aminoacids themselves. In certain embodiments, the changes involveconservative amino acid substitutions. Conservative amino acidsubstitution may involve replacing one amino acid with another that has,e.g., similar hydrorphobicity, hydrophilicity, charge, or aromaticity.In certain embodiments, conservative amino acid substitutions may bemade on the basis of similar hydropathic indices. A hydropathic indextakes into account the hydrophobicity and charge characteristics of anamino acid, and in certain embodiments, may be used as a guide forselecting conservative amino acid substitutions. The hydropathic indexis discussed, e.g., in Kyte et al., J. Mol. Biol., 157:105-131 (1982).It is understood in the art that conservative amino acid substitutionsmay be made on the basis of any of the aforementioned characteristics.

Alterations to the amino acids may include, but are not limited to,glycosylation, methylation, phosphorylation, biotinylation, and anycovalent and noncovalent additions to a protein that do not result in achange in amino acid sequence. “Amino acid” as used herein refers to anyamino acid, natural or nonnatural, that may be incorporated, eitherenzymatically or synthetically, into a polypeptide or protein.

As used herein, an “affinity set” is a set of molecules thatspecifically bind to one another. Affinity sets include, but are notlimited to, biotin and avidin, biotin and streptavidin, receptor andligand, antibody and ligand, antibody and antigen, and a polynucleotidesequence and its complement. In certain embodiments, affinity sets thatare bound may be unbound. For example, a polynucleotide sequences thatare hybridized may be denatured, and biotin bound to streptavidin may beheated and become unbound.

A “target” refers to any material that can be distinguished by a probe.Targets may include both naturally occurring and synthetic molecules.

In certain embodiments, targets may include nucleic acid sequences. Incertain embodiments, target nucleic acid sequences may include RNA andDNA. Exemplary RNA target sequences include, but are not limited to,mRNA, rRNA, tRNA, viral RNA, and variants of RNA, such as splicingvariants. Exemplary DNA target sequences include, but are not limitedto, genomic DNA, plasmid DNA, phage DNA, nucleolar DNA, mitochondrialDNA, and chloroplast DNA.

In certain embodiments, nucleic acid sequences include, but are notlimited to, cDNA, yeast artificial chromosomes (YAC's), bacterialartificial chromosomes (BAC's), other extrachromosomal DNA, and nucleicacid analogs. Exemplary nucleic acid analogs include, but are notlimited to, LNAs, PNAs, PPG's, and other nucleic acid analogs discussedbelow.

A variety of methods are available for obtaining a target nucleic acidsequence for use with the compositions and methods of the presentinvention. When the nucleic acid target is obtained through isolationfrom a biological matrix, certain isolation techniques include (1)organic extraction followed by ethanol precipitation, e.g., using aphenol/chloroform organic reagent (e.g., Ausubel et al., eds., CurrentProtocols in Molecular Biology Volume 1, Chapter 2, Section I, JohnWiley & Sons, New York (1993)), preferably using an automated DNAextractor, e.g., the Model 341 DNA Extractor available from PE AppliedBiosystems (Foster City, Calif.); (2) stationary phase adsorptionmethods (e.g., Boom et al., U.S. Pat. No. 5,234,809; Walsh et al.,Biotechniques 10(4): 506-513 (1991)); and (3) salt-induced DNAprecipitation methods (e.g., Miller et al., Nucleic Acids Research,16(3): 9-10 (1988)), such precipitation methods being typically referredto as “salting-out” methods. In certain embodiments, the above isolationmethods may be preceded by an enzyme digestion step to help eliminateunwanted protein from the sample, e.g., digestion with proteinase K, orother like proteases.

In certain embodiments, target nucleic acid sequences include, but arenot limited to, amplification products, ligation products, transcriptionproducts, reverse transcription products, primer extension products,methylated DNA, and cleavage products. In certain embodiments, thetarget nucleic acid sequences may be produced by whole genomeamplification. In certain embodiments, the target nucleic acid sequencesmay be produced by isothermal amplification and/or ligation.

In certain embodiments, nucleic acids in a sample may be subjected to acleavage procedure such as the cleavage procedure in an Invader™ assay(as exemplified, e.g., in U.S. Pat. Nos. 5,846,717; 5,985,557;5,994,069; 6,001,567; and 6,090,543). Such procedures produce a cleavageproduct when a nucleic acid of interest is present in a sample. Incertain embodiments, the target may be such a cleavage product. Briefly,the cleavage procedure may employ two nucleic acid oligonucleotides thatare designed to be complementary to the nucleic acid in the sample. Afirst oligonucleotide comprises a 5′ portion that does not complementthe nucleic acid in the sample, contiguous with a 3′ portion that doescomplement the nucleic acid in the sample. A second oligonucleotidecomplements the nucleic acid in the sample in a region of the nucleicacid in the sample that is 3′ of the region complemented by the firstoligonucleotide, and includes a complementary or non-complementaryportion that slightly overlaps with the region complemented by the firstoligonucleotide. Hybridization of the two oligonucleotides to thenucleic acid in the sample causes a portion of the first oligonucleotideto be cleaved, often in the presence of an enzyme. The cleavage productis typically the 5′ portion of the first oligonucleotide that does notcomplement the nucleic acid in the sample, and that portion of thecomplementary region that overlaps with the second oligonucleotide. Thiscleavage product comprises a known nucleic acid sequence. In certainembodiments, such cleavage products may be targets.

Different target nucleic acid sequences may be different portions of asingle contiguous nucleic acid or may be on different nucleic acids.Different portions of a single contiguous nucleic acid may overlap.

In certain embodiments, a target nucleic acid sequence comprises anupstream or 5′ region, a downstream or 3′ region, and a “pivotalnucleotide” located between the upstream region and the downstreamregion (see, e.g., FIG. 1). The pivotal nucleotide is the nucleotidebeing detected by the probe set and may represent, for example, withoutlimitation, a single polymorphic nucleotide in a multiallelic targetlocus.

The person of ordinary skill will appreciate that while a target nucleicacid sequence is typically described as a single-stranded molecule, theopposing strand of a double-stranded molecule comprises a complementarysequence that may also be used as a target sequence.

Other targets include, but are not limited to, peptide sequences.Peptides sequences include, but are not limited to, proteins, fragmentsof proteins, and other segments of amino acids. In certain embodiments,peptide target sequences include, but are not limited to, differentpeptide alleles (similar peptides with different amino acids) anddifferent peptide conformations (similar proteins with differentsecondary and tertiary structures). Other naturally occurring targetsinclude, but are not limited to, hormones and other signal molecules,such as hormones and other steroid-type molecules.

In certain embodiments, targets include, but are not limited to,synthetic peptides, pharmaceuticals, and other organic small molecules.

Probes

The term “probe” or “target-specific probe” is any moiety that comprisesa portion that can specifically bind a target. Probes may include, butare not limited to, nucleic acids, peptides, and other molecules thatcan specifically bind a target in a sample. Such specific bindingincludes, but is not limited to, hybridization between nucleic acidmolecules, antibody-antigen interactions, interactions between ligandsand receptors, and interactions between aptomers and proteins.

In certain embodiments, a probe comprises a nucleic acidsequence-specific portion that is designed to hybridize in asequence-specific manner with a complementary region on a selectedtarget nucleic acid sequence. In certain embodiments, thesequence-specific portion of the probe may be specific for a particularsequence, or alternatively, may be degenerate, e.g., specific for a setof sequences. A probe for a target peptide may comprise an antibody, asa non-limiting example.

In certain embodiments, probes comprise aptomers, which are nucleicacids that specifically bind to certain peptide sequences. In certainembodiments, probes comprise peptides. Such peptides include, but arenot limited to, antibodies and receptor molecules. In certainembodiments, probes comprise antibodies directed to specific targetpeptide antigens.

In certain embodiments, probes may include other members of uniquebinding pairs, such as streptavidin/biotin binding pairs, and affinitybinding chemicals available from Prolinx™ (Bothell, Wash.) asexemplified, e.g., by U.S. Pat. Nos. 5,831,046; 5,852,178; 5,859,210;5,872,224; 5,877,297; 6,008,406; 6,013,783; 6,031,17; and 6,075,126.

A “probe set” according to the present invention is a group of two ormore probes designed to detect at least one target. As a non-limitingexample, a probe set may comprise two nucleic acid probes designed tohybridize to a target such that, when the two probes are hybridized tothe target adjacent to one another, they are suitable for ligationtogether.

When used in the context of the present invention, “suitable forligation” refers to at least one first target-specific probe and atleast one second target-specific probe, each comprising an appropriatelyreactive group. Exemplary reactive groups include, but are not limitedto, a free hydroxyl group on the 3′ end of the first probe and a freephosphate group on the 5′ end of the second probe, phosphorothioate andtosylate or iodide, esters and hydrazide, RC(O)S⁻, haloalkyl, RCH₂S andα-haloacyl, thiophosphoryl and bromoacetoamido groups, andS-pivaloyloxymethyl-4-thiothymidine. Additionally, in certainembodiments, the first and second target-specific probes are hybridizedto the target sequence such that the 3′ end of the first target-specificprobe and the 5′ end of the second target-specific probe are immediatelyadjacent to allow ligation.

Codeable Labels

The term “label” refers to any molecule or set of molecules that canprovide a detectable signal or interacts with a second molecule or othermember of the set of molecules to provide a detectable signal—eitherprovided by the first molecule or provided by the second molecule, e.g.,FRET (Fluorescent Resonance Energy Transfer). Use of labels can beaccomplished using any one of a large number of known techniquesemploying known labels, linkages, linking groups, reagents, reactionconditions, and analysis and purification methods. Labels include, butare not limited to, light-emitting or light-absorbing compounds whichgenerate or quench a detectable fluorescent, chemiluminescent, orbioluminescent signal (see, e.g., Kricka, L. in Nonisotopic DNA ProbeTechniques (1992), Academic Press, San Diego, pp. 3-28). Fluorescentreporter dyes useful as labels include, but are not limited to,fluoresceins (see, e.g., U.S. Pat. Nos. 5,188,934; 6,008,379; and6,020,481), rhodamines (see, e.g., U.S. Pat. Nos. 5,366,860; 5,847,162;5,936,087; 6,051,719; and 6,191,278), benzophenoxazines (see, e.g., U.S.Pat. No. 6,140,500), energy-transfer fluorescent dyes, comprising pairsof donors and acceptors (see, e.g., U.S. Pat. Nos. 5,863,727; 5,800,996;and 5,945,526), and cyanines (see, e.g., Kubista, WO 97/45539), as wellas any other fluorescent moiety capable of generating a detectablesignal. Examples of fluorescein dyes include, but are not limited to,6-carboxyfluorescein; 2′,4′,1,4,-tetrachlorofluorescein; and2′,4′,5′,7′,1,4-hexachlorofluorescein.

Other exemplary labels include, but are not limited to, luminescentmolecules that emit light, and molecules that can be involved inluminescent reactions, such as luciferin-luciferase reactions, as anon-limiting example. Labels also include, but are not limited to,chemiluminescent and electroluminescent molecules and reactions. As anon-limiting example, chemiluminescent labels may be exposed to film.Development of the film indicates whether or not targets are present inthe sample or the quantity of the targets in the sample.

Other exemplary labels include, but are not limited to, donor-acceptorinteractions, in which a donor molecule emits energy that is detected byan acceptor molecule. The acceptor molecule then emits a detectablesignal.

Other exemplary labels include, but are not limited to, molecules thatare involved in infrared photon release.

Labels also include, but are not limited to, quantum dots. “Quantumdots” refer to semiconductor nanocrystalline compounds capable ofemitting a second energy in response to exposure to a first energy.Typically, the energy emitted by a single quantum dot always has thesame predictable wavelength. Exemplary semiconductor nanocrystallinecompounds include, but are not limited to, crystals of CdSe, CdS, andZnS. Suitable quantum dots according to certain embodiments aredescribed, e.g., in U.S. Pat. Nos. 5,990,479 and 6,207,392 B1, and in“Quantum-dot-tagged microbeads for multiplexed optical coding ofbiomolecules,” Han et al., Nature Biotechnology, 19:631-635 (2001).

Labels of the present invention also include phosphors andradioisotopes. Radioisotopes may be directly detected, or may excite afluorophore that emits a wavelength of light that is then detected.Phosphor particles may be excited by an infrared light (approximatelyaround 980 nm) but emit signals within the visible spectrum, thussignificantly reducing or eliminating background light.

Other examples of certain exemplary labels include particles with codedinformation, such as barcodes, and also include the microparticle tagsdescribed in U.S. Pat. No. 4,053,433. Certain other non-radioactivelabeling methods, techniques, and reagents are reviewed in:Non-Radioactive Labelling, A Practical Introduction, Garman, A. J.(1997) Academic Press, San Diego.

A class of labels effect the separation or immobilization of a moleculeby specific or non-specific capture, for example biotin, digoxigenin,and other haptens (see, e.g., Andrus, A. “Chemical methods for 5′non-isotopic labeling of PCR probes and primers” (1995) in PCR 2: APractical Approach, Oxford University Press, Oxford, pp. 39-54).

“Codeable label” refers to the one or more labels which is specific to aparticular moiety. In certain embodiments the moiety is a target and/ora probe. In embodiments in which a codeable label comprises more thanone label, the labels may be the same or different. Detection of a givencodeable label indicates the presence of the moiety to which thecodeable label is specific. The absence of a given codeable labelindicates the absence of the moiety to which the codeable label isspecific.

Codeable labels may be described as “detectably different,” which meansthat they are distinguishable from one another by at least one detectionmethod. Different codeable labels include, but are not limited to, oneor more labels that emit light of different wavelengths, one or morelabels that emit light of different intensities, one or more labels thatemanate different numbers and/or patterns of signals, one or more labelsthat have different fluorescent decay lifetimes, one or more labels thathave different spectral signatures, one or more labels that havedifferent radioactive decay properties, one or more labels of differentcharge, and one or more labels of different size.

In certain embodiments, the number of codeable labels is counted, whichrefers to the actual counting of individual codeable labels. Countingthe number of codeable labels is distinguishable from analog signaldetection, where an aggregate level of signal from multiple labels isdetected. Analog signal detection typically uses integration of signalsfrom multiple labels of the same type to determine the number of suchlabels present in a sample. For example, analog detection typicallyprovides an estimate of the number of labels of a given type bycomparing the brightness or level of intensity of the signal in the testsample to the brightness or level of intensity of the signal in controlswith known quantities of the given labels.

Counting, by contrast, is a digital detection system in which the numberof individual codeable labels is actually counted. Thus, in certainembodiments, if 200 of the same codeable labels are present in a sample,each of those labels is actually counted. In certain embodiments, thenumber of labels counted may be within 20% of the actual number in thesample. In certain embodiments, the number of labels counted may bewithin 10% of the actual number in the sample. In certain embodiments,the number of labels counted may be within 50% of the actual number inthe sample. In certain embodiments, a representative portion of thoselabels present in a sample are counted, and the total number of labelsin the sample is determined by the number of labels counted in therepresentative portion. In contrast, to determine the number of labelsin a sample with analog detection, the aggregate signal from the 200labels is measured and compared to the aggregate signal from knownquantities of labels.

In certain embodiments, the codeable labels and probes in a reaction arein sufficient excess of the target available that the number of codeablelabels counted is representative to the number of targets present. Insuch embodiments, there is typically no more than one target bound toeach codeable label.

In certain embodiments, since it involves the actual counting ofcodeable labels, digital detection may be less influenced by background“noise,” or incidental light that may be interpreted as part of theaggregate signal in analog detection.

In certain embodiments, one may determine fine distinctions betweendifferent numbers of codeable labels in different samples by countingthe number of codeable labels. In contrast, the aggregate signal frommultiple labels in analog detection, in certain instances, may beaffected by the variable amount of background signal in differentsamples, which may obscure small differences in the number of labels indifferent samples.

In certain embodiments where two or more detectably different codeablelabels are being detected in a sample, possible inaccuracies due tooverlapping signals from detectably different codeable labels may beminimized by counting each of the detectably different codeable labels.In certain analog detection methods, part of the signal from one labelmay be detected as signal from another different label, which may resultin an inaccurate reading. This may be particularly the case if thesignals from the different labels have overlapping emission ranges. Bycounting the individual codeable labels, in certain embodiments,inaccuracies that may sometimes result may be minimized from analogdetection where one measures the aggregate signal intensities fromdifferent labels.

In certain embodiments, the “codeable labels” are different sets ofquantum dots that are specific for different target-specific probes (thedifferent probes being specific for different target sequences), and thedifferent sets of quantum dots are detectably different from oneanother.

Codeable labels may be attached directly to probes, or indirectlyattached to other molecules that are then attached to probes. In certainembodiments, the codeable labels may be attached to a probe prior tobeing added to a sample, or may become attached to a probe during thecourse of a reaction that forms a detectable complex. In certainembodiments, codeable labels may be attached directly to a probe, orthrough a linking molecule, such as a chemical linkage group, or linkingpair, such as a streptavidin-biotin pair.

In certain embodiments, labels are incorporated into beads, which maythen be attached to probes. A “bead” refers to any material to whichprobes can be attached. Beads may be of any shape, including, but notlimited to, spheres, rods, cubes, and bars. Beads may be made of anysubstance, including, but not limited to, silica glass and polymers.Beads may be any size. Certain non-limiting examples of beads includethose described, e.g., in U.S. Pat. Nos. 4,499,052 (Fulwyler); 4,717,655(Fulwyler); 3,957,741 (Rembaum, CalTech); 4,035,316 (Rembaum, CalTech);4,105,598 (Rembaum, CalTech); 4,224,198 (Rembaum, CalTech); 4,326,008(Rembaum, CalTech); 3,853,987 (Dreyer, CalTech); 4,108,972 (Dreyer,CalTech); 5,093,234 (Flow Cytometry Standards); 6,268,222 (Luminex);5,326,692 (Molecular Probes); 5,573,909 (Molecular Probes); 5,723,218(Molecular Probes); 5,786,219 (Molecular Probes); 5,028,545 (Soini); and5,132,242 (Sau Cheung); as well as international application PublicationNos. WO 01/13119 (Luminex); WO 01/14589 (Luminex); WO 97/14028(Luminex); WO 99/19515 (Luminex); WO 99/37814 (Luminex); WO 99/52708(Luminex); WO 00/55363 (Amersham); WO 01/01141 (Amersham); WO 99/64867(Amersham); and WO 94/11735 (Soini).

In certain embodiments, the beads comprise coated or uncoated particlescomprising at least one of magnetic material, paramagnetic material,silica glass, polyacrylamide, polysaccharide, plastic, latex,polystyrene, and other polymeric substances.

Beads may comprise codeable labels, such as sets of quantum dotsaccording to certain embodiments. Those skilled in the art are aware ofsuitable methods of obtaining beads with quantum dots. See, e.g., Han etal., Nature Biotechnology, 19:631-635 (2001), and U.S. Pat. Nos.6,207,392 (Shuming Nie); 6,114,038 (Biocrystal); 6,261,779 (Biocrystal);6,207,229 (Bawendi); 6,251,303 (Bawendi); 6,274,323 (Quantum Dot);5,990,479 (Alivisatos); 6,207,392 (Alivisatos); internationalapplication Publication Nos. WO 00/29617 (Shuming Nie); WO 00/27365(Biocrystal); WO 00/28089 (Biocrystal); WO 01/89585 (Biocrystal); WO00/17642 (Bawendi); WO 00/17656 (Bawendi); WO 99/26299 (Bawendi); WO00/68692 (Quantum Dot); WO 00/55631 (Alivisatos); and EuropeanApplication No. 0 990 903 A1 (Bawendi). The quantum dots or other labelsmay be embedded in beads.

In certain embodiments, as a non-limiting example, quantum dots may beincorporated into cross-linked polymer beads. In certain embodiments,polystyrene beads may be synthesized using an emulsion of styrene (98%vol./vol.), divinylbenzene (1% vol./vol.), and acrylic acid (1%vol./vol.) at 70° C. In certain embodiments, the beads are then swelledin a solvent mixture containing 5% (vol./vol.) chloroform and 95%(vol./vol.) propanol or butanol. In certain embodiments, a controlledamount of ZnS-capped CdSe quantum dots are added to the mixture. Afterincubation at room temperature, the embedding process is complete. Incertain embodiments, the size of the beads may be controlled by theamount of a stabilizer (e.g., polyvinylpyrrolidone) used in thesynthesis. In certain embodiments, a spherical bead 2 μm in diametercontaining quantum dots that are 2-4 nm in diameter may contain tens ofthousands of quantum dots.

The method of manufacturing beads discussed above may result in beadswith varying numbers of quantum dots. Also, if one uses more than onecolor of quantum dot, one may obtain beads that have varying numbers ofthe different colors. In certain embodiments, after such beadpreparation, the resulting beads are sorted by the relative number ofquantum dots of each color in a given bead to obtain groups ofidentically labeled beads with distinct codeable labels. In certainembodiments, the sorting can be automated by machines, such as aFluorescence Associated Cell Sorter (FACS) or other flow-cytometer typedetection method that can distinguish between different codeable labels.

One of skill will appreciate that there are many methods of obtainingbeads comprising probes. Such methods include, but are not limited to,attaching the probes to the beads using covalent bonding, UVcrosslinking, and linking through an affinity set. As a non-limitingexample, streptavidin molecules may be covalently attached to thecarboxylic acid groups on the bead surface. Oligonucleotide probes maybe biotinylated, then linked to the beads via the streptavidinmolecules.

In certain embodiments, a bead contains an internal reference label. Incertain embodiments, the internal reference label is detectablydifferent than the codeable label. In certain embodiments, one may usean internal reference label to confirm the number of beads with codeablelabels. For example, in certain embodiments, beads with differentcodeable labels will each include the same internal reference label thatcan be used to identify the presence of a single bead. In certainembodiments, in order to distinguish a single first bead with a codeablelabel from two beads with codeable labels that have a combined intensitysimilar to the intensity of the codeable label of the first bead, asingle internal reference label in each bead may be included. In certainembodiments, detection of two internal reference labels would indicatethe presence of two beads, while detection of a single internalreference label would indicate the presence of a single bead. Thus, incertain embodiments, internal reference labels assist in accuratedetermination of the number of beads actually present when detection ofcodeable labels alone may provide ambiguous results.

For example, in certain embodiments in which a bead comprises codingelements comprising fluorophores, dyes, or nanocrystals, the internalreference label may be a single quantum dot in each bead. The presenceof a single quantum dot may be used to indicate the presence of a singlebead. The presence of two quantum dots would indicate the presence oftwo beads, and so forth.

As another nonlimiting example, in certain embodiments, an internalreference label may provide a color signal that is detectably differentfrom the signal of the codeable labels. In certain embodiments, thesignal from the internal reference label for each bead will have anintensity that can be used to identify the presence of a single bead.For example, in certain embodiments, the internal reference signal foreach bead will provide a red signal with an intensity of about one unit.In certain such embodiments, one may employ two different codeablelabels on two different beads to detect two different targets. Forexample, in certain embodiments the first codeable label for a firsttarget provides a green signal having an intensity of one unit, and thesecond codeable label for a′ second target provides a green signalhaving an intensity of two units. Without an internal reference label,in certain embodiments, one may have difficulty determining whether agreen signal having an intensity of two units indicates the presence oftwo beads for the first target or the presence of one bead for thesecond target. In certain embodiments that employ the red internalreference label, the detection of a red signal with an intensity of oneunit will indicate the presence of one bead for the second target, andthe detection of a red signal with an intensity of two units willindicate the presence of two beads for the first target.

When beads of varying size are employed, the amount of label, such as afluorescent dye as a non-limiting example, incorporated into such beadsmay vary according to the size of the bead. In certain embodiments, theinclusion of an internal reference label in beads may be used tonormalize variations in codeable labels signal caused by variations inbead size.

In certain embodiments that do not employ an internal reference label,one tries to use beads of fairly uniform size to try to avoiddifferences in signal from the same codeable label due to the differencein the sizes of the beads. In certain embodiments, an internal referencelabel on the beads may permit one to use beads of varying size. Incertain such embodiments, one may employ two different codeable labelson two different beads to detect two different targets. For example, ifthe beads have a diameter of X, the first codeable label for a firsttarget provides a green signal having an intensity of one unit, and thesecond codeable label for a second target provides a green signal havingan intensity of two units. Without an internal reference label, incertain embodiments with beads of varying size, one may have difficultydetermining whether a bead providing a green signal having an intensityof two units indicates the presence of a bead for the first targethaving a diameter larger than X or the presence of a bead for the secondtarget having a diameter X.

In certain such embodiments, one may employ beads that include aninternal reference label that is detectably different from the codeablelabels. In certain embodiments, one may employ an internal referencelabel that provides a red signal having an intensity of one unit if thebead has a diameter of X. Thus, if the bead size varies from thediameter of X, the internal reference label will provide a differentintensity than one unit. In certain such embodiments, the detection of abead with a green signal of two units indicates the presence of thesecond target if the red signal is one unit and indicates the presenceof the first target if the red signal is two units.

In certain embodiments, the use of an internal reference label may allowone to produce beads of smaller sizes than is practical without the useof an internal reference label. In certain embodiments, beads may beless than 2 μm in diameter. In certain embodiments, using an internalreference label, one may be able to distinguish very small differencesin bead size.

In certain embodiments, the use of an internal reference label could beused when counting beads by staging or by flow cytometry. In certainembodiments, beads employing an internal reference label may be used inan array, wherein analytes are bound to specific regions of the array.In certain embodiments, arrays with beads with internal reference labelsmay be imaged. In certain embodiments, software may be used to normalizesignals using the internal reference labels in digitalized images.

In certain embodiments, the size of a bead may be used as a codingelement. As a non-limiting example, beads have 100 different codesemploying two colors. In certain embodiments, different sized beads maybe used as part of the code, because different sized beads providedifferent intensities. For example, in certain embodiments, the 100codes using two colors may be increased to 400 codes by using fourdifferent sized beads.

Detectable Complexes

The term “detectable complex” of the present invention is a complexcomprising codeable label. In certain embodiments, a detectable complexfurther comprises at least one probe.

According certain embodiments, a detectable complex is produced when atarget is present and is not produced when a target is absent. Incertain embodiments, a detectable complex is formed if the target andprobe specifically bind one another.

In certain embodiments, the detectable complex is produced in a ligationreaction. Ligation methods include, but are not limited to, bothenzymatic and chemical ligation.

A ligation reaction according to the present invention comprises anyenzymatic or chemical process wherein an internucleotide linkage isformed between the opposing ends of nucleic acid sequences that areadjacently hybridized to a template. Additionally, the opposing ends ofthe annealed nucleic acid sequences typically are suitable for ligation(suitability for ligation is a function of the ligation methodemployed). The internucleotide linkage may include, but is not limitedto, phosphodiester bond formation. Such bond formation may include,without limitation, those created enzymatically by a DNA or RNA ligase,such as bacteriophage T4 DNA ligase, T4 RNA ligase, Thermus thermophilus(Tth) ligase, Thermus aquaticus (Taq) ligase, or Pyrococcus furiosus(Pfu) ligase. Other internucleotide linkages include, withoutlimitation, covalent bond formation between appropriate reactive groupssuch as between an α-haloacyl group and a phosphothioate group to form athiophosphorylacetylamino group, a phosphorothioate a tosylate or iodidegroup to form a 5′-phosphorothioester, and pyrophosphate linkages.

Chemical ligation agents include, without limitation, activating,condensing, and reducing agents, such as carbodiimide, cyanogen bromide(BrCN), N-cyanoimidazole, imidazole,1-methylimidazole/carbodiimide/cystamine, dithiothreitol (DTT) andultraviolet light. Autoligation, i.e., spontaneous ligation in theabsence of a ligating agent, is also within the scope of the invention.Detailed protocols for chemical ligation methods and descriptions ofappropriate reactive groups can be found, among other places, in Xu etal., Nucleic Acid Res., 27:875-81 (1999); Gryaznov and Letsinger,Nucleic Acid Res. 21:1403-08 (1993); Gryaznov et al., Nucleic Acid Res.22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry 25:7423-30 (1986);Luebke and Dervan, Nucleic Acids Res. 20:3005-09 (1992); Sievers and vonKiedrowski, Nature 369:221-24 (1994); Liu and Taylor, Nucleic Acids Res.26:3300-04 (1999); Wang and Kool, Nucleic Acids Res. 22:2326-33 (1994);Purmal et al., Nucleic Acids Res. 20:3713-19 (1992); Ashley and Kushlan,Biochemistry 30:2927-33 (1991); Chu and Orgel, Nucleic Acids Res.16:3671-91 (1988); Sokolova et al., FEBS Letters 232:153-55 (1988);Naylor and Gilham, Biochemistry 5:2722-28 (1966); and U.S. Pat. No.5,476,930.

In certain embodiments, one may employ at least one cycle of thefollowing sequential procedures: hybridizing the sequence-specificportions of a first target-specific probe and a second target-specificprobe, that are suitable for ligation, to their respective complementarytarget regions; ligating the 3′ end of the first target-specific probewith the 5′ end of the second target-specific probe to form a ligationproduct; and denaturing the nucleic acid duplex to separate the ligationproduct from the target sequence. The cycle may or may not be repeated.For example, without limitation, by thermocycling the ligation reactionto linearly increase the amount of ligation product.

Also within the scope of the invention are ligation techniques such asgap-filling ligation, including, without limitation, gap-filling OLA andLCR, bridging oligonucleotide ligation, and correction ligation.Descriptions of these techniques can be found, among other places, inU.S. Pat. No. 5,185,243, published European Patent Applications EP320308 and EP 439182, and published PCT Patent Application WO 90/01069.

Detectable complexes may also be produced by hybridization of nucleicacids without any ligation steps. In certain embodiments, hybridizationoccurs with PNA, LNA, or other synthetic nucleic acids that have ahigher Tm than naturally occurring nucleic acid hybridizations.

Other detectable complexes also may be produced by antibody-antigeninteractions, aptomer-protein interactions, and action of other specificbinding pairs (e.g., streptavidin-biotin reactions).

Detectable complexes may also be produced by primer extension reactions.Primer extension reactions include, but are not limited to, single baseextension (SBE) reactions, sequencing reactions (for example Sangerdideoxy sequencing reactions), and other reactions including polymerase.

In certain embodiments, the detectable complex is produced in aligand-receptor reaction. As a non-limiting example, a codeable labeland a probe may be attached to the ligand molecule. The receptor isattached to a separating moiety, such as a magnetic bead.

In certain embodiments, a probe is hybridized to a target nucleic acidsequence, and a codeable label bound to a single nucleotide is attachedto the probe by a polymerase reaction when the target nucleic acid ispresent.

In certain embodiments, a probe comprising a nucleic acid, complementaryto a nucleic acid target sequence, is attached to a separating moietysuch as a magnetic bead. In certain embodiments, the probe is then addedto a sample containing the nucleic acid target sequence. In certainembodiments, codeable labels attached to nucleotides are added to thesample with a polymerase. In certain embodiments, if the nucleic acidtarget sequence is present in the sample, the probe hybridizes to thetarget, and the polymerase adds the codeable label-attached nucleotidesto the oligonucleotide probe, forming a detectable complex. In certainembodiments, after denaturation from the sample nucleic acids, theprobes are then separated from the sample using the magnetic beads. Incertain embodiments, if a detectable complex is counted, then a targetis present in the sample.

In certain embodiments, the number of targets in a sample is representedby the number of detectable complexes removed from a sample as comparedto the number of detectable complexes present at the start of adetection reaction. In certain embodiments, a number of detectablecomplexes are added to a sample. In certain embodiments, thesedetectable complexes comprise a codeable label attached to one end of asingle-stranded nucleic acid probe, and a separating moiety attached tothe other end of the single-stranded nucleic acid probe. In certainembodiments, when a target is present in the sample, the targethybridizes to the single-stranded probe to form a double strandedmolecule. In certain embodiments, an endonuclease is added to thereaction which cuts double-stranded nucleic acid. In such embodiments,the number of detectable complexes remaining is equal to the number ofdetectable complexes initially added to the reaction less the number oftargets present in the sample.

Separating Moieties and Methods

The term “separating moiety” refers to any moiety that, when included ina detectable complex, may be used to separate the detectable complexfrom at least one other moiety in the sample.

In certain embodiments, separation is achieved without any particularseparating moiety incorporated in a detectable complex. In certainembodiments, methods that do not employ a specific separating moietyinclude, but are not limited to, separation based on density, size,electrical or ionic charge, diffusion, heat, flow cytometry, anddirected light. In certain embodiments, the detection of detectablecomplexes occurs without any separation of detectable complexes fromother moieties.

In certain embodiments, the methods comprise separating the detectablecomplex from separating moieties that are not in a detectable complex,prior to the quantitating, or the detecting the presence or absence of,one or more targets. One of ordinary skill will appreciate that thereare several methods that may be used according to certain embodimentsfor separating detectable complexes from separating moieties not in adetectable complex. As non-limiting examples in certain embodiments,differences in density or size of separating moieties may be used toseparate detectable complexes from separating moieties not in adetectable complex. Methods of separation include, but are not limitedto, use of sizing filters, sizing columns, density gradients, separationby gravity, and separation by centrifugation. Examples of suchsize-separating moieties include, but are not limited to, polymer beads.

For example, in certain embodiments, one may separate detectablecomplexes from separating moieties not in a detectable complex asfollows. Probe sets may include a separating bead comprising a firstprobe and a detecting bead comprising a second probe. The separatingbead further comprises a first codeable label, and the detecting beadfurther comprises a second codeable label. The separating beads aresmaller in size than the detecting beads. After ligation, one canseparate detectable complexes from separating beads based on thedifferences in sizes of the detecting beads in the detectable complexesand the separating beads not in detectable complexes. For example, onemay pass the material through a sizing filter that allows separatingbeads to flow through and which retains detectable complexes.

Also, in certain embodiments, one may separate detectable complexes fromseparating moieties not in detectable complexes as follows. Probe setsmay include a separating bead comprising a first probe and a detectingbead comprising a second probe. The separating bead further comprises afirst codeable label, and the detecting bead further comprises a secondcodeable label. The separating beads have a higher density than thedetecting beads. After a detectable complex is formed, one can separatedetectable complexes from separating beads not in a detectable complex,based on the differences in density of the detecting beads in thedetectable complexes and the free separating beads. For example, incertain embodiments, one may place the material in a density gradient,which will separate the detectable complexes from the free separatingbeads. Also, in certain embodiments, gravity may be used to separate thedetectable complexes from the free separating beads. Thus, if theseparating beads have a higher density than the detecting beads, theseparating beads will sink below the detectable complexes.

According to certain embodiments, other different properties of theprobes in a probe set may be used to separate detectable complexes fromprobes and codeable labels not in detectable complexes. For example, incertain embodiments, one may use a separating moiety that has aparticular property that attracts it to a particular position and othermoieties in the reaction mixture that lack that property. For example,according to certain embodiments, the separating moiety may comprise amagnetic particle and the other moieties in the reaction mixture do notcomprise a magnetic particle.

The term “magnetic particle” refers to material which can be moved usinga magnetic force. This includes, but is not limited to, particles thatare magnetized, particles that are not magnetized but are influenced bymagnetic fields (e.g., colloidal iron, iron oxides (e.g., ferrite andmagnetite), nickel, and nickel-iron alloys), and particles which canbecome magnetized (e.g., ferrite, magnetite, iron, nickel, and alloysthereof).

In certain embodiments, the magnetic particle comprises one or more offerrite, magnetite, nickel, and iron, and the other moieties in thereaction mixture do not comprise such a material. In such embodiments,one can use such distinctive properties of the separating moiety toseparate detectable complexes that include a separating bead from probesand codeable labels not in detectable complexes.

Other methods of separating detectable complexes from other moietiesinclude, but are not limited to, separation by density, separation byelectrical charge, separation by drag coefficiencies (e.g.,electrophoretic mobility), separation by diffusion or dialysis, andseparation by heat or light (e.g., employing lasers to move labeledparticles).

In certain embodiments, one may remove separating moieties, codeablelabels, or probes not in detectable complexes (free components) from acomposition containing detectable complexes prior to the quantitatingthe target nucleic acid sequence or sequences in the sample. In certainembodiments, one may remove detectable complexes from a compositioncontaining free components prior to the quantitating (or detecting thepresence or absence of) the target nucleic sequence or sequences in thesample.

In certain embodiments, separating the detectable complex from freecomponents comprises separating the detectable complex from the targetnucleic acid sequence, and separating the detectable complex from thesample.

In certain embodiments, the detectable complex is a ligation product. Incertain embodiments, separating of the detectable complex from thetarget sequence comprises thermal denaturation.

As a nonlimiting illustration, in certain embodiments, one may detectthe presence or absence of different target nucleic acid sequences in asample, such as a cell lysate, as follows. A sample is combined with adifferent probe set specific for each of the different target nucleicacid sequences. Each probe set comprises a separating bead comprising amagnetic particle incorporated into a bead and a first target-specificprobe and comprises a detecting bead comprising a bead and a secondtarget-specific probe. The separating beads of the probe sets have ahigher density than the detecting beads.

The separating bead of each probe set further comprises a first codeablelabel that is specific for the first target-specific probe, and thedetecting bead of each bead set further comprises a second codeablelabel that is specific for the second target-specific probe. The firstcodeable label is detectably different from the second codeable label.The target-specific probes in each bead set are suitable for ligationtogether when hybridized adjacent to one another on a complementarytarget sequence.

When the sample includes a complementary target nucleic acid sequence tothe first and second target-specific probes of a given probe set, theprobes anneal and are ligated in the presence of ligase (L) to form adetectable complex comprising the separating bead and the detecting bead(see, e.g., FIG. 7). After ligation, the target nucleic acid sequence isthermally denatured from the detectable complex.

In certain embodiments depicted in FIGS. 8 and 9, the sample is thensubjected to a density gradient such that detectable complexes anddetecting beads are situated above separating beads in the vessel.Detectable complexes may then be separated from unligated detectingbeads by a magnetic source (see FIG. 8). For example, one can remove thedetectable complexes from the sample containing the unligated detectingbeads using the magnetic source, and can place the detectable complexesin a separate vessel that does not contain any unligated beads (seeFIGS. 8 and 9). One can then detect the presence or absence ofdetectable complexes by counting of the unique combinations of codeablelabels.

In certain embodiments, one may use a second magnet near the bottom ofthe vessel, which will attract and hold the higher density unligatedseparating beads but will not attract or hold the lower densitydetectable complexes and unligated detecting beads (see FIG. 8). Such asecond magnet near the bottom of the vessel, however, is not mandatory.For example, in certain embodiments, one can design the density of thebeads such that distance of separation between any unligated separatingbeads and the detectable complex allows attraction of the detectablecomplex to a magnetic device and does not allow attraction of theunligated separating beads to the magnetic device.

In certain embodiments depicted in FIGS. 10 and 11, after ligation, thesample is heated to denature the hybridized probes and target nucleicacid sequences. Due to gravity, the unligated separating beads sinkbelow the detectable complexes and unligated detecting beads (see FIG.10). One may then place an electro-magnet (magnet) near the bottom ofthe vessel such that it attracts and holds the higher density unligatedseparating beads, and such that it does not attract or hold the lowerdensity detectable complexes and unligated detecting beads (see FIG.11). Also, one may place an electro-magnet (magnet) into the top of thevessel such that it attracts and holds detectable complexes (see FIG.11).

The electro-magnet holding the detectable complexes may then be liftedto separate the detectable complexes from the unligated detecting beads(see FIGS. 11 and 12). One can then detect the presence or absence ofdetectable complexes, without removing them from the sample includingthe unligated beads, by counting the unique combinations of codeablelabels (see FIG. 12). FIG. 12 depicts certain embodiments wheredetectable complexes are illuminated, the codes are identified, andligation products are counted with a PMT sensor.

In certain embodiments depicted in FIG. 18, detectable complexes areformed comprising a magnetic bead comprising a first codeable label anda nonmagnetic bead comprising a second codeable label. In certainembodiments, an electromagnet (magnet) is placed beneath a reactionvessel containing beads and detectable complexes. When the electromagnetis turned on, detectable complexes and magnetic beads that are not in adetectable complex are attracted to the bottom of the vessel. See FIG.18 C.

In certain embodiments, nonmagnetic beads that are not in a detectablecomplex and other nonmagnetic moieties are removed by a continuous flowsystem, comprising an input tube and an output tube. See FIG. 18D. Incertain embodiments, the electromagnet is then turned off, and thedetectable complexes and the magnetic beads that are not in a detectablecomplex are then pulled out with a flow cytometer tube. See FIG. 18E.

In certain embodiments, the detectable complexes and the magnetic beadsthat are not in a detectable complex are then sent through a flowcytometer and only combinations of the first and second codeable labelsare counted. See FIG. 18F. In such embodiments, the magnetic beads thatare not in a detectable complex will include only a first codeablelabel, which will not be counted.

In certain embodiments, one may carry out the method discussed above forFIG. 18 with a magnetic bead that does not include a codeable label.After separation of the nonmagnetic beads that are not in a detectablecomplex, the detectable complexes and the magnetic beads that are not ina detectable complex are then sent through a flow cytometer. Since themagnetic beads do not have codeable label in such embodiments, only thecodeable labels of the nonmagnetic beads in the detectable complexes arecounted.

In certain embodiments depicted in FIG. 19, detectable complexes areformed comprising a magnetic bead, a nonmagnetic bead, and a codeablelabel. In certain embodiments, a first electromagnet is placed beneath areaction vessel containing beads and detectable complexes. When thefirst electromagnet is turned on, detectable complexes and magneticbeads that are not in a detectable complex are attracted to the bottomof the vessel. See FIG. 19 C.

In certain embodiments, nonmagnetic beads that are not in a detectablecomplex and other nonmagnetic moieties are removed by a continuous flowsystem, comprising an input tube and an output tube. See FIG. 19D. Incertain embodiments, a vessel is used that may be inverted such that asubstantial amount of liquid will not drain out when it is inverted. Incertain embodiments, this may be accomplished using a small vessel inwhich surface tension inhibits drainage of liquid out of the vessel whenthe vessel is inverted. In embodiments that employ an inverted vessel,the vessel and the first electromagnet are then inverted and the firstelectromagnet is turned off. A second electromagnet is then turned on atthe bottom of the inverted vessel to attract the detectable complexesand the magnetic beads that are not in a detectable complex. See FIG.19E. In certain embodiments, the detectable complexes have more drag andless density than the magnetic beads that are not in a detectablecomplex. Thus, in such embodiments, the magnetic beads that are not in adetectable complex move faster than the detectable complexes toward thesecond electomagnet. After the magnetic beads that are not in adetectable complex are collected onto the second electromagnet (see FIG.19E), the vessel is inverted back before the detectable complexes havereached the second electromagnet.

In certain embodiments, the detectable complexes are then pulled outwith a flow cytometer tube (see FIG. 19F), and are sent through a flowcytometer and the codeable labels of the detectable complexes arecounted.

In certain embodiments, one may carry out the method discussed above forFIG. 19 with a magnetic bead that does not include a codeable label. Incertain embodiments, one may carry out the method discussed above forFIG. 19 with a nonmagnetic bead that does not include a codeable label.

In certain embodiments depicted in FIG. 20, detectable complexes areformed comprising a magnetic bead, a nonmagnetic bead, and a codeablelabel. A filter is included in the vessel. In certain embodiments, themagnetic beads are designed such they can pass through the filter andthe nonmagnetic beads are designed such that they cannot pass throughthe filter. In certain embodiments, an electromagnet is placed beneaththe reaction vessel containing beads and detectable complexes. When thefirst electromagnet is turned on, detectable complexes and magneticbeads that are not in a detectable complex are attracted to the bottomof the vessel. See. FIG. 20 C.

The magnetic beads that are not in a detectable complex pass through thefilter toward the magnet. The detectable complexes are pulled toward themagnet, but cannot pass through the filter in view of the nonmagneticbead of the complex. The detectable complexes are held at the filter bythe pull of the magnet. In certain embodiments, nonmagnetic beads thatare not in a detectable complex and other nonmagnetic moieties are thenremoved by a continuous flow system, comprising an input tube and anoutput tube. See FIG. 20D. In certain embodiments, detectable complexescan then be separated from magnetic beads that are not in a detectablecomplex by moving the filter away from the electromagnet. Such movementof the filter pulls the detectable complexes away from the electromagnetand away from the magnetic beads that are not in a detectable complex.

In certain embodiments, detectable complexes are then pulled out with aflow cytometer tube. See FIG. 20E. In certain embodiments, thedetectable complexes are then sent through a flow cytometer and thecodeable labels of the detectable complexes are counted. See FIG. 20 F.

In certain embodiments, one may carry out the method discussed above forFIG. 20 with a magnetic bead that does not include a codeable label. Incertain embodiments, one may carry out the method discussed above forFIG. 20 with a nonmagnetic bead that does not include a codeable label.

In certain embodiments, one may use grooves in a vessel that help toalign detectable complexes in a manner that facilitates the detection ofthe presence or absence of sets of labels. In certain embodiments,“aligned ligation products” are products in which the separating beadsof the products are closer to a given surface of a vessel than thedetecting beads. For example, in certain embodiments depicted in FIG.13, the separating beads may be smaller in size than the detectingbeads. A groove is designed such that the separating beads fit into thegroove, and the separating beads are too large to fit into the groove(see FIG. 13). In certain embodiments, one may place a magnetic sourcenear the groove in the vessel to attract and hold separating beads intothe groove. One can then count the combinations of codeable labels todetect the presence or absence of detectable complexes. In certainembodiments shown in FIG. 13, the grooves position the detectablecomplexes such that an angled excitation beam illuminates both beadswith a few readings.

In certain embodiments, electrophoresis may also be used to separateseparating moieties by charge or by a charge:mass ratio. In certainembodiments, a charged separating moiety may also be separated by ionexchange, e.g., by using an ion exchange column or a charge-basedchromatography.

In certain embodiments, a separating moiety may also be a member of anaffinity set. An affinity set is a set of molecules that specificallybind to one another. Exemplary affinity sets include, but are notlimited to, strepavidin-biotin pairs, complementary nucleic acids,antibody-antigen pairs, and affinity binding chemicals available fromProlinx™ (Bothell, Wash.) as exemplified by U.S. Pat. Nos. 5,831, 046;5,852,178; 5,859,210; 5,872,224; 5,877,297; 6,008,406; 6,013,783;6,031,17; and 6,075,126.

In certain embodiments, separating moieties are separated in view oftheir mobility. In certain embodiments, separating in view of mobilityis accomplished by the size of the separating moiety. In certainembodiments, mobility modifiers may be employed during electrophoresis.Exemplary mobility modifiers and methods of their use have beendescribed, e.g., in U.S. Pat. Nos. 5,470,705; 5,580,732; 5,624,800; and5,989,871. In certain embodiments, by changing the mobility of acodeable label, one may distinguish signals associated with the presenceof a target from signals from labels not associated with the presence ofa target.

In certain embodiments, two or more different separating moieties ormethods may be used. As a nonlimiting example, in certain embodiments, adetectable complex may comprise a magnetic bead, a ligation product, anda biotin-coated bead. See, e.g., FIG. 21, part A. A streptavidin-coatedelectromagnet is placed in the sample and turned on See, e.g., FIG. 21,part B. The detectable complexes and the magnetic beads that are not ina detectable complex are attracted to the electromagnet. Thebiotin-coated beads in the detectable complexes bind to the streptavidinon the electromagnet. See, e.g., FIG. 21, part C. The electromagnet isthen turned off, and the magnetic beads that are not in a detectablecomplex fall off the electromagnet, while the detectable complexesremain bound to the electromagnet. In certain embodiments, theelectromagnet is then removed from the sample with the detectablecomplexes bound to the electromagnet, and the codeable labels in thedetectable complexes are detected by camera or scanner. See, e.g., FIG.21, part D.

In certain embodiments, the presence of a target prevents, rather thanfacilitates, the formation of a detectable complex. In certain suchembodiments, the number of probes and/or codeable labels are limited. Incertain such embodiments, counting of detectable complexes provides anumber of codeable labels that are not associated with targets.Subtracting the number of detectable complexes that are counted from thetotal number of detectable complexes expected in the complete absence oftargets provides the number of targets present in the sample.

Tube in Tube Separation of Detectable Complexes

In certain embodiments depicted in FIG. 28, detectable complexes areformed comprising a bead comprising a codeable label and a separatingmoiety, such as a biotin molecule. In certain embodiments, one mayinclude a ligation reaction in the process of forming detectablecomplexes. In certain embodiments, one may include an antibody-peptidereaction in the process of forming detectable complexes. In certainembodiments, separating moieties other than biotin may be employed.

In the embodiments depicted in FIG. 28, a probe set is employed thatincludes a first probe with a bead comprising a codeable label and asecond probe attached to biotin. In FIG. 28 A, ligation occurs if thetarget is present to form the detectable complex. In certain embodimentsshown in FIG. 28(B), streptavidin-coated magnetic beads are added. Thestreptavidin-coated magnetic beads bind to biotin molecules in thedetectable complexes. In certain embodiments, the beads comprisingcodeable labels that are not ligated to biotin molecules do not bind tothe streptavidin-coated magnetic beads. In certain embodiments, theprocess shown in FIG. 28 may be modified by employing a process thatdoes not involve ligation to form a detectable complex comprising a beadcomprising a codeable label and biotin.

As shown in certain embodiments depicted in FIG. 28(C), a first tube isprovided that is placed within a second tube which is larger. In certainembodiments, the two tubes are partially filled with a buffer that has adensity greater than the beads comprising a codeable label. In certainembodiments, the second tube is partially filled with the buffer, andthe second tube is placed in the outer tube, such that the buffer comesto an equilibrium height in the first and second tubes.

In certain embodiments, after the ligation reaction, at least a portionof the sample is placed on top of the high density buffer inside thefirst tube, such that the beads float in the buffer. In certainembodiments depicted in FIG. 28(D), a magnet is then applied to thebottom of the second tube such that unbound streptavidin-coated magneticbeads and detectable complexes are attracted to the magnet at the bottomof the second tube. Beads comprising codeable labels not in a detectablecomplex remain floating on or toward the top of the high density bufferwithin the first tube. In certain embodiments depicted in FIG. 28(E),the top of the first tube is substantially sealed sealed, and the firsttube is lifted above the buffer in the second tube. Thus, the beadscomprising codeable labels not in detectable complexes are separatedfrom the buffer containing the detectable complexes. In certainembodiments, the first tube prevents beads from sticking to the walls ofthe second tube.

In certain embodiments, the detectable complexes in the second tube maythen be removed to be counted. In certain embodiments, the detectablecomplexes are subjected to a using a flow cytometer. In certainembodiments, the magnet on the bottom of the second tube is removed withthe detectable complexes, and the codeable labels are detected on themagnet.

Detection Methods

In certain embodiments, the present invention provides for the detectionof codeable labels. In certain embodiments, the present inventionprovides for the counting of labels. Several methods of label detectionand/or counting are envisioned, and one of skill in the art willappreciate the variety of methods by which one could detect and/or countcodeable labels of the present invention.

As discussed above, counting of codeable labels refers to the actualcounting of individual labels. In certain embodiments, detection and/orcounting further includes identifying the code of a label if multipledetectably different labels are employed in the same procedure.

In certain embodiments, codeable labels are detected with a type of flowcytometry, such as a Fluorescence Associated Cell Sorter (FACS), aLuminex™ detection device, or a similar technology developed for thedetection of single codeable label molecules. In certain embodiments,codeable labels are resolved by electrophoresis and detected during orafter electrophoretic migration of the codeable labels. Electrophoresisincludes, but is not limited to, capillary electrophoresis and fieldelectrophoresis. In certain embodiments, such methods involve a devicethat excites the codeable labels (such as a laser, as a non-limitingexample) and a scanning device that counts the codeable labels. Incertain embodiments shown in FIG. 9, detectable complexes are releasedinto a detection vessel and detectable complexes that settle to thebottom of the vessel are read by a PMT sensor. In certain embodiments,the PMT sensor is a six element PMT device with ten 500 kHz digitizersthat scan 10 million beads in under 60 minutes.

In certain embodiments, other methods of detection involve staticmethods of detection. In certain embodiments, such methods involveplacing the codeable labels or complexes on a plate (as a non-limitingexample), exciting the codeable labels with one or more excitationsources (such as lasers or different wavelengths, for example) andrunning a scanning device across the plate in order to count thecodeable labels. In certain embodiments, the plate is moved back andforth across the field of detection of the scanning device. In certainembodiments, the codeable labels are attached to the plate or slide. Incertain embodiments, a camera could image the entire field, and theimage could be scanned in order to count the codeable labels.

According to certain embodiments, multiple targets may be detected in asample, and distinguished by using different codeable labels. In certainembodiments, the codeable labels can be coded using two or more labels(e.g., in certain embodiments, quantum dots, fluorophores, or dyes areused). In certain embodiments, one may use multiple wavelengths orcolors of labels, which multiplies the number of potential differentcodeable labels. For example, if a given codeable label is given abinary code, then one can detect the presence or absence of a specificcolor of label (either a “1” or “0”—hence a binary code). If only onebinary color is used, then there are 2 codes, one with the label, andone without the label. If two binary colors are used (e.g., red andblue), then 4 codes are possible—(1) red, (2) blue, (3) red and blue,and (4) no color (see, e.g., FIG. 3). Each additional color multipliesthe number of possible codes by two. Thus, if 10 colors of labels areused, 1,024 binary codes are possible.

In certain embodiments, the codeable labels are incorporated or attachedto beads. In certain embodiments, the codeable labels may be attacheddirectly to probes without being incorporated into beads.

Intensity may also be used as a factor in distinguishing codeablelabels. In certain embodiments, intensity variations may be accomplishedusing codeable labels that include the same number of labels of a singlewavelength, but different codeable labels with different probes havelabels with different intensity levels. In certain embodiments,intensity variations may be accomplished using codeable labels thatinclude the same number of labels of a single emission spectrum, butdifferent codeable labels with different probes have labels withdifferent intensity levels. In certain embodiments, intensity variationsmay be accomplished by varying the number of labels of the samewavelength in different codeable labels attached to different probes. Incertain embodiments, intensity variations may be accomplished by varyingthe number of labels of the same emission spectrum in different codeablelabels attached to different probes. For example, in certainembodiments, one can use labels of the same wavelength in differentcodeable labels, and distinguish between the codeable labels usingdifferent numbers of labels in each different set. For example, if acodeable label is given a ternary code (three levels of intensity foreach color of label), then one color of label provides three possiblecodes —(1) no label, (2) one label, and (3) two labels. If two colorsare used, then 9 ternary codes are possible (see the nonlimiting examplein FIG. 3). Six colors would allow 729 ternary codes.

Further, when codeable labels are attached to two probes of a probe set(for example, by incorporation into beads), the number of potentialcodes is further multiplied (see the non-limiting examples in FIG. 4).For example, using two colors in a binary code, 16 different probe setcodes are possible (4×4). Using two colors in a ternary code, 81different probe set codes are possible (9×9). Using 10 colors in abinary code, over 1 million probe set codes are possible (1,024×1,024).Using 6 colors in a ternary code, over 500,000 probe set codes arepossible (729×729).

In addition, in certain embodiments, the labels, such as quantum dotsfor example, are particularly efficient in transmitting a signal suchthat codeable label can be detected. In certain such embodiments, thecodeable labels may be used to detect very few molecules within a samplewithout target amplification.

In certain embodiments, a probe set comprises a separating bead thatcomprises a separating moiety, a first probe, and a first codeablelabel; and comprises a detecting bead that comprises a second probe anda second codeable label. In certain such embodiments, the first codeablelabel has a level of intensity that is specific for the first probe. Incertain such embodiments, the second codeable label has a level ofintensity that is specific for the second probe. In certain embodiments,the beads of a probe set comprise labels of the same wavelength, but thefirst codeable label has a level of intensity that is specific for thefirst probe, and the second codeable label has a level of intensity thatis specific for the second probe.

In certain embodiments, the codeable label comprises at least 1,000labels, wherein the labels have predetermined wavelength combinationsthat make each codeable label distinguishable from other codeablelabels.

In certain embodiments, the codeable labels may comprise any number oflabels from two to over 1,000. In certain embodiments, one uses codeablelabels that allow one to detect the presence or absence of particulartarget-specific probes in a detectable complex. In certain embodiments,one uses codeable labels such that the detection of the presence of aparticular combination of labels confirms the presence of one specificdetectable complex. And, the detection of the absence of such aparticular combination of labels confirms the absence of that onespecific detectable complex.

In certain embodiments, the labels are selected from quantum dots,phosphors, and fluorescent dyes.

In certain embodiments that employ a first bead and a second bead, boththe separating bead and detecting bead of the probe sets comprise amagnetic particle. In certain such embodiments, the beads are elongatedand comprise a magnetic particle on one end and a target-specific probeon the other end. The beads further comprise labels placed in aparticular order along the length of the bead (see, e.g., FIG. 14( a)).See, e.g., U.S. Pat. No. 4,053,433, which describes elongated polymerswith labels in particular orders.

In certain embodiments, the polarity or orientation of the magneticparticles in the beads is designed to facilitate alignment of thedetectable complexes. For example, in certain embodiments, the vesselcontaining the detectable complexes will include a groove on a surfacethat is placed near a magnetic source (see, e.g., FIG. 14( b)). Thebeads are designed so that the polarity or orientation of the magneticparticles in the beads results in the detectable complexes aligning inthe groove with the first bead of each detectable complex closer to oneend of the groove than the second bead of that detectable complex (see,e.g., FIG. 14( b)). One can then quantify detectable complexes byquantitating the particular order of combinations of codeable labels.

In such embodiments, one can use a probe set that has a first bead and asecond bead that comprise identical codeable labels, since the order ofthe identical codeable labels will be different on the first bead and onthe second bead in the aligned detectable complexes (see, e.g., FIG. 14(c)).

Exemplary Embodiments of the Invention

In certain embodiments in which the targets are nucleic acid sequences,the sequence-specific portions of the probes are of sufficient length topermit specific annealing to complementary sequences in targetsequences. In certain embodiments, the length of the sequence-specificportion is 12 to 35 nucleotides. Detailed descriptions of probe designthat provide for sequence-specific annealing can be found, among otherplaces, in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual,Cold Spring Harbor Press, 1995, and Kwok et al. (Nucl. Acid Res.18:999-1005, 1990).

In certain embodiments, a probe set according to the present inventioncomprises a first target-specific probe and a second target-specificprobe that adjacently hybridize to the same target sequence. Asequence-specific portion of the first target-specific probe in eachprobe set is designed to hybridize with the downstream region of thetarget sequence in a sequence-specific manner (see, e.g., probe A inFIG. 1). A sequence-specific portion of the second target-specific probein the probe set is designed to hybridize with the upstream region ofthe target sequence in a sequence-specific manner (see, e.g., probe Z inFIG. 1). The sequence-specific portions of the probes are of sufficientlength to permit specific annealing with complementary sequences intarget sequences, as appropriate. Under appropriate conditions,adjacently hybridized probes may be ligated together to form a ligationproduct, provided that they comprise appropriate reactive groups, forexample, without limitation, a free 3′-hydroxyl or 5′-phosphate group.

In certain embodiments, two different probe sets may be used toquantitate two different target sequences that differ by one or morenucleotides (see, e.g., FIG. 2). According to certain embodiments of theinvention, a probe set is designed so that the sequence-specific portionof the first target-specific probe will hybridize with the downstreamtarget region (see, e.g., probe A in FIG. 1, and probes A and B in FIG.2) and the sequence-specific portion of the second target-specific probewill hybridize with the upstream target region (see, e.g., probe Z inFIG. 1 and FIG. 2). In certain embodiments, a nucleotide basecomplementary to the pivotal nucleotide, the “pivotal complement,” ispresent on the proximal end of either the first target-specific probe orthe second target-specific probe of the probe set (see, e.g., 3′ end ofprobe A in FIG. 1, and the 3′ end of probes A and B in FIG. 2).

When the first and second target-specific probes of the probe set arehybridized to the appropriate upstream and downstream target regions,and the pivotal complement is base-paired with the pivotal nucleotide onthe target sequence, the hybridized first and second target-specificprobes may be ligated together to form a ligation product (see, e.g.,FIG. 2( b)-(c)). A mismatched base at the pivotal nucleotide, however,interferes with ligation, even if both probes are otherwise fullyhybridized to their respective target regions (see, e.g., FIG. 2(b)-(c)). Thus, in certain embodiments, highly related sequences thatdiffer by as little as a single nucleotide can be distinguished.

For example, according to certain embodiments, one can distinguish thetwo potential alleles in a biallelic locus using two different probesets as follows. The first target-specific probe of each probe set willdiffer from one another in their pivotal complement, and the codeablelabels associated with the two different first target-specific probeswill be detectably different (see, e.g., the codeable labels with probesA and B in FIG. 2( a)) Each probe set can also comprise identical secondtarget-specific probes, and the codeable labels associated with the twoidentical second target-specific probes will be identical (see, e.g.,the codeable labels with probe Z in FIG. 2( a)).

One can combine the sample with the two different probe sets. (Incertain embodiments, one of the probes of each probe set can furthercomprise a separating moiety. All three target-specific probes willhybridize with the target sequence under appropriate conditions (see,e.g., FIG. 2( b)). Only the first target-specific probe with thehybridized pivotal complement, however, will be ligated with thehybridized second target-specific probe (see, e.g., FIG. 2( c)). Thus,if only one allele is present in the sample, only one ligation productfor that target will be generated (see, e.g., ligation product A-Z inFIG. 2( d)). Both ligation products (A-Z and B-Z) would be formed in asample from a heterozygous individual.

Further, in certain embodiments, probe sets do not comprise a pivotalcomplement at the terminus of the first or the second target-specificprobe. Rather, the target nucleotide or nucleotides to be detected arelocated within the sequence-specific portion of either the firsttarget-specific probe or the second target-specific probe. Probes withsequence-specific portions that are fully complementary with theirrespective target regions will hybridize under high stringencyconditions. Probes with one or more mismatched bases in thesequence-specific portion, by contrast, will not hybridize to theirrespective target region. Both the first target-specific probe and thesecond target-specific probe must be hybridized to the target for aligation product to be generated. The nucleotides to be detected may beboth pivotal or internal.

In certain embodiments, the first target-specific probes and secondtarget-specific probes in a probe set are designed with similar meltingtemperatures (T_(m)). In certain embodiments, where a probe includes apivotal complement, the T_(m) for the probe(s) comprising the pivotalcomplement(s) of the target pivotal nucleotide sought will beapproximately 4-6° C. lower than the other probe(s) that do not containthe pivotal complement in the probe set. The probe comprising thepivotal complement(s) will also preferably be designed with a T_(m) nearthe ligation temperature. Thus, in such embodiments, a probe with amismatched nucleotide will more readily dissociate from the target atthe ligation temperature. In such embodiments, the ligation temperature,therefore, provides another way to discriminate between, for example,multiple potential alleles in the target.

Certain Exemplary Embodiments of Detecting Targets

The present invention is directed to methods, reagents, and kits forquantitating targets in a sample. In certain embodiments, one detectsthe presence or absence of target nucleic acid sequences using ligation.

In certain embodiments, for each target nucleic acid sequence to bedetected, a probe set, comprising at least one first target-specificprobe and at least one second target-specific probe, is combined withthe sample and optionally, a ligation agent, to form a ligation reactionmixture. The at least one first probe further comprises a first codeablelabel comprising at least two labels, and the first codeable label isspecific for the first target-specific probe. The at least one secondprobe further comprises a second codeable label comprising at least twolabels, and the second codeable label is specific for the secondtarget-specific probe. The first codeable label is detectably differentfrom the second codeable label.

In certain embodiments, the first and second target-specific probes ineach probe set are designed to be complementary to the sequencesimmediately flanking the pivotal nucleotide of the target sequence (see,e.g., probes A, B, and Z in FIG. 2( a)). Either the firsttarget-specific probe or the second target-specific probe of a probeset, but not both, will comprise the pivotal complement (see, e.g.,probe A of FIG. 2( a)). When the target sequence is present in thesample, the first and second target-specific probes will hybridize,under appropriate conditions, to adjacent regions on the target (see,e.g., FIG. 2( b)). When the pivotal complement is base-paired in thepresence of an appropriate ligation agent, two adjacently hybridizedprobes may be ligated together to form a ligation product (see, e.g.,FIG. 2( c)).

One can then detect the presence or absence of the target nucleic acidsequences by detecting the presence or absence of the ligation product.

In certain embodiments, including, but not limited to, detectingmultiple alleles, the ligation reaction mixture may comprise a differentprobe set for each potential allele in a multiallelic target locus. Incertain embodiments, one may use, for example, without limitation, asimple screening assay to detect the presence of three biallelic loci(e.g., L1, L2, and L3) in an individual using six probe sets. See, e.g.,Table 1 below.

TABLE 1 Locus Allele Probe Set - Probe (label) L1 1 A (2 red), Z (2blue) 2 B (4 red), Z (2 blue) L2 1 C (2 orange), Y (4 blue) 2 D (4orange), Y (4 blue) L3 1 E (2 yellow), X (2 green) 2 F (4 yellow), X (2green)

In such embodiments, two different probe sets are used to detect thepresence or absence of each allele at each locus. The two firsttarget-specific probes of the two different probe sets for each locus,for example, probes A and B for locus L1, comprise the same upstreamsequence-specific portion, but differ at the pivotal complement. Also,the two different probes A and B comprise different codeable labels. Thetwo second target-specific probes of the two different probe sets foreach locus, for example, probe Z for locus L1, comprise the samedownstream sequence-specific portion. Also, the probes Z comprise thesame codeable label. (In certain embodiments, one of the probes of eachprobe set may further comprise a separating moiety, and the other probeof each probe set may not comprise a separating moiety.)

Thus, in embodiments as depicted in Table 1, three probes A, B, and Z,are used to form the two possible L1 ligation products, wherein AZ isthe ligation product of the first L1 allele and BZ is the ligationproduct of the second L1 allele. Likewise, probes C, D, and Y, are usedto form the two possible L2 ligation products. Likewise, probes E, F,and X, are used to form the two possible L3 ligation products.

After ligation of adjacently hybridized first and second target-specificprobes, one can detect the presence or absence of a ligation product foreach of the alleles for each of the loci by detecting the presence ofabsence of the unique combinations of codeable labels for each allele.For example, one may detect the following combinations of codeablelabels: (1) 2 red/2 blue; (2) 4 orange/4 blue; (3) 2 yellow/2 green; and(4) 4 yellow/2 green. Such an individual would be determined to behomozygous for allele 1 at locus L1, homozygous for allele 2 at locusL2, and heterozygous for both alleles 1 and 2 at locus L3.

The person of ordinary skill will appreciate that in certainembodiments, three or more alleles at a multiallelic locus can also bedifferentiated using these methods. Also, in certain embodiments, morethan one loci can be analyzed.

The skilled artisan will understand that in certain embodiments, theprobes can be designed with the pivotal complement at any location ineither the first target-specific probe or the second target-specificprobe. Additionally, in certain embodiments, target-specific probescomprising multiple pivotal complements are within the scope of theinvention.

Detection of Splice Variants

According to certain embodiments, the present invention may be used toidentify splice variants in a target nucleic acid sequence. For example,genes, the DNA that encodes for a protein or proteins, may contain aseries of coding regions, referred to as exons, interspersed bynon-coding regions referred to as introns. In a splicing process,introns are removed and exons are juxtaposed so that the final RNAmolecule, typically a messenger RNA (mRNA), comprises a continuouscoding sequence. While some genes encode a single protein orpolypeptide, other genes can code for a multitude of proteins orpolypeptides due to alternate splicing.

For example, a gene may comprise five exons each separated from theother exons by at least one intron, see FIG. 5. The hypothetical genethat encodes the primary transcript, shown at the top of FIG. 5, codesfor three different proteins, each encoded by one of the three maturemRNAs, shown at the bottom of FIG. 5. Due to alternate splicing, exon 1may be juxtaposed with (a) exon 2a-exon 3, (b) exon 2b-exon 3, or (c)exon 2c-exon 3, the three splicing options depicted in FIG. 5, whichresult in the three different versions of mature mRNA.

The rat muscle protein, troponin T is but one example of alternatesplicing. The gene encoding troponin T comprises five exons (W, X, α, β,and Z), each encoding a domain of the final protein. The five exons areseparated by introns. Two different proteins, an α-form and a β-form areproduced by alternate splicing of the troponin T gene. The α-form istranslated from a mRNA that contains exons W, X, α, and Z. The β-form istranslated from a mRNA that contains exons W, X, β, and Z.

In certain embodiments, a method is provided for detecting the presenceor absence of different splice variants in at least one target nucleicacid sequence in a sample using a different probe set for each differentsplice variant.

Certain nonlimiting embodiments for identifying splice variants areillustrated by FIG. 6. Such embodiments permit one to identify twodifferent splice variants. One splice variant includes exon 1, exon 2,and exon 4 (splice variant E1 E2E4). The other splice variant includesexon 1, exon 3, and exon 4 (splice variant E1 E3E4).

The probe set that is specific for splice variant E1 E2E4 comprises atleast one first target-specific probe Q that comprises asequence-specific portion that hybridizes to at least a portion of exon1, e.g., it can hybridize to the end of exon 1 that is adjacent toeither exon 2 or exon 3. The at least one first target specific probefurther comprises a first codeable label. The probe set that is specificfor splice variant E1 E2E4 further comprises at least one secondtarget-specific probe R that comprises a sequence-specific portion thathybridizes to at least a portion of exon 2, e.g., it can hybridize tothe end of exon 2 that is adjacent to exon 1. The at least one secondtarget-specific probe of the probe set that is specific for splicevariant E1 E2E4 further comprises a second codeable label that isdetectably different from the first codeable label.

The probe set that is specific for splice variant E1 E3E4 comprises atleast one first probe that is the same as the at least one first probeof the probe set that is specific for the splice variant E1 E2E4: Theprobe set that is specific for splice variant E1 E3E4 further comprisesat least one second target-specific probe S that comprises asequence-specific portion that hybridizes to at least a portion of exon3, e.g., it can hybridize to the end of exon 3 that is adjacent toexon 1. The at least one second probe of the probe set that is specificfor splice variant E1 E3E4 further comprises a second codeable labelthat is detectably different from the first codeable label and isdetectably different from the second codeable label of the at least onesecond probe of the probe set that is specific for the splice variant E1E2E4.

After ligation of adjacently hybridized first and second target-specificprobes, one can detect the presence or absence of a ligation product foreach of the splice variants by detecting the presence or absence of theunique combinations of codeable labels for each splice variant. Forexample, in certain methods depicted in FIG. 6, if the presence of aligation product with the combination of two dots and four dots isdetected, the presence of splice variant E1 E2E4 in the sample isdetected. If that combination of codeable labels is absent, the absenceof splice variant E1 E2E4 in the sample is detected. Also, in certainmethods depicted in FIG. 6, if the presence of a ligation product withthe combination of two dots and six dots is detected, the presence ofsplice variant E1 E3E4 in the sample is detected. If that combination ofcodeable labels is absent, the absence of splice variant E1 E3E4 in thesample is detected.

In certain embodiments, the at least one target nucleic acid sequencecomprises at least one complementary DNA (cDNA) generated from an RNA.In certain embodiments, the at least one cDNA is generated from at leastone messenger RNA (mRNA). In certain embodiments, the at least onetarget nucleic acid sequence comprises at least one RNA target sequencepresent in the sample.

Methods Employing Addressable Portions

In certain embodiments, one employs unique specifically addressableoligonucleotides, or “addressable portions.” Addressable portions areoligonucleotide sequences designed to hybridize to the complement of theaddressable portion. For a pair of addressable portions that arecomplementary to one another, one member will be called an addressableportion and the other will be called a complementary addressableportion.

In certain embodiments, the method comprises forming a ligation mixturecomprising a first probe, comprising a first addressable portion and afirst target-specific portion; a second probe, comprising a secondaddressable portion and a second target-specific portion, wherein thefirst and second target-specific portions are suitable for ligationtogether when hybridized adjacent to one another on a target; a ligationagent; and a sample. If a target is present in the sample, the first andsecond target-specific portions of the first and second probes areligated together to form a ligation product.

FIG. 22 illustrates exemplary embodiments which include a ligationreaction mixture comprising: a first probe 12 that comprises a firsttarget-specific portion 14 and a first addressable portion 18; and asecond probe 20 that comprises a second target-specific portion 22 and asecond addressable portion 24. The target-specific portions of theprobes hybridize to a target 16. The ligation reaction mixture issubjected to a ligation reaction, and the first and secondtarget-specific portions of the first and second probes are ligatedtogether to form a ligation product.

In certain embodiments, after the formation of any ligation productscomprising a first addressable portion and second addressable portion,bead pairs are added to the ligation mixture. In certain embodiments, abead pair comprises a first bead comprising a first complementaryaddressable portion, wherein the first complementary addressable portionis complementary to the first addressable portion; and a second beadcomprising a second complementary addressable portion, wherein thesecond complementary portion is complementary to the second addressableportion. The first addressable portion of the ligation producthybridizes to the first complementary addressable portion of the firstbead, and the second addressable portion of the ligation producthybridizes to the second complementary addressable portion of the secondbead, to form a detectable complex comprising the first bead, theligation product, and the second bead. In certain embodiments, eitherthe first bead or the second bead or both beads may be separationmoieties. In certain embodiments, either the first bead or the secondbead or both beads may comprise a codeable label.

In certain embodiments, the complementary addressable portion associatedwith a bead is included in a hairpin structure. In certain embodiments,the hairpin structure comprises a complementary addressable portion andan anchor portion. In certain embodiments, the anchor portion isupstream from the complementary addressable portion, and the anchorportion comprises a first portion and a second portion that complementeach other. In certain embodiments, the anchor portion of the hairpinstructure is attached to the bead. Certain exemplary embodiments ofhairpin structures 52 and 54 are shown in FIG. 23.

In certain embodiments, the first and second portions of the anchorportion of the hairpin structure hybridize to one another such that theanchor portion includes one end that is contiguous with thecomplementary addressable portion and an opposite free end that foldsback onto the end contiguous with the complementary addressable portion.See, e.g., FIG. 23. In certain embodiments, a first addressable portionand a second addressable portion of a ligation product hybridize to afirst complementary addressable portion and a second complementaryaddressable portion, respectively, that are included in two differenthairpin structures that are attached to two different beads to form adetectable complex. See, e.g., FIG. 23. In certain embodiments, thehairpin structures are designed such that the free end of the anchorportion is suitable for ligation together with an adjacent addressableportion of a ligation product that is hybridized to the hairpinstructure. See, e.g., FIG. 23. In certain such embodiments, the ligationproduct and the hairpin structure are subjected to a ligation reaction.In such embodiments, the detectable complex comprises the beads and theligation product that is ligated to the hairpin structures attached tothe beads.

In certain embodiments, after the formation of any ligation productscomprising a first addressable portion and second addressable portion,linking oligonucleotide pairs and bead pairs are added to the ligationmixture. Certain such exemplary embodiments are shown in FIG. 24. Incertain embodiments, a bead pair comprises a first bead comprising athird addressable portion; and a second bead comprising a fourthaddressable portion. In such embodiments, the linking oligonucleotidepair comprises a first linking oligonucleotide and a second linkingoligonucleotide. The first linking oligonucleotide comprises: a firstcomplementary addressable portion that is complementary to the firstaddressable portion of the ligation product; and a third complementaryaddressable portion that is complementary to the third addressableportion of the first bead. The second linking oligonucleotide comprises:a second complementary addressable portion that is complementary to thesecond addressable portion of the ligation product; and a fourthcomplementary addressable portion that is complementary to the fourthaddressable portion of the second bead. The first, second, third, andfourth specific addressable portions hybridize to the first, second,third, and fourth specific addressable portions, respectively, to form adetectable complex comprising the ligation product and the beads. Incertain embodiments, either the first bead or the second bead or bothbeads may be separation moieties. In certain embodiments, either thefirst bead or the second bead or both beads may comprise a codeablelabel.

In certain embodiments, the linking oligonucleotides are designed suchthat after hybridization of the first, second, third, and fourthspecific addressable portions to the first, second, third, and fourthspecific addressable portions, respectively, adjacent ends of the firstand third addressable portions are suitable for ligation together, andadjacent ends of the second and fourth addressable portions are suitablefor ligation together. In certain such embodiments, the hybridizedligation product, linking oligonucleotides, and addressable portions ofthe beads are subjected to a ligation reaction. In such embodiments, thedetectable complex comprises the beads and the ligation product that isligated to the addressable portions of the beads.

In certain embodiments, after the formation of any ligation productscomprising a first addressable portion and second addressable portion,the ligation products are separated from excess probes that are nothybridized to a target. In certain such embodiments, one may employ afilter that is designed such that it captures target and such thatunhybridized probes pass through it. Since the ligatation products arehybridized to target, the ligation products will also be captured by thefilter. In certain embodiments, one removes the probes that are nothybridized to the target and proceeds with the ligation products.

In certain embodiments, one can separate ligation products from targetsby denaturing the ligation product from the target. In certainembodiments, the target can then be destroyed. For example, in certainembodiments in which the target is mRNA, Rnase can be added to decomposethe mRNA without damaging the ligation products.

Single-Bead Assay

In certain embodiments, the detection of a target may be carried outusing two separating moieties and one codeable label. In certainembodiments, the detection uses a probe set comprising a firstoligonucleotide probe comprising a first addressable portion (Z1) and afirst target-specific portion (TSO1), as shown in FIG. 25. In certainembodiments, the probe set further comprises a second oligonucleotideprobe comprising a second target specific portion (TSO2), a non-specificspacer portion (Z1), and first separating moiety, such as a biotinmolecule, as shown in FIG. 25. In certain embodiments, in the presenceof the target, the first target-specific portion and the secondtarget-specific portion hybridize to the target such that the twotarget-specific portions are suitable for ligation. Thus, in certainembodiments, if a target molecule is present, two adjacently hybridizedfirst and second ligation probes may be ligated together to form aligation product comprising the first and second target-specificportions, the first addressable portion, the spacer portion, and thebiotin molecule, as shown in FIG. 25.

In certain embodiments, the method employs a bead comprising a codeablelabel that is attached to one or more oligonucleotides comprising asecond addressable portion (Z2). See, e.g., FIG. 26. In certainembodiments, the second addressable portion Z2 is complementary to thefirst addressable portion Z1 such that, if a ligation product is formed,the probe set forms a detectable complex comprising the bead comprisingthe codeable label, the second addressable portion Z2 hybridized to thefirst addressable portion Z1, the first and second target-specificportions (TSO1 and TSO2), the spacer portion (S), and the biotinmolecule, as shown in FIG. 26.

In certain embodiments, the oligonucleotide comprising the secondaddressable portion Z2 is covalently attached to the bead comprising thecodeable label. In certain embodiments, the second addressable portionZ2 may comprise DNA, LNA, PNA, 2′-O-methyl nucleic acid, or any otherprobe material. In certain embodiments, the sequences of the first andsecond addressable portions (Z1 and Z2) are optimized to a particularmelting temperature or hybridization binding strength.

In certain embodiments, the second addressable portion Z2 is part of ahairpin structure attached to the bead comprising the codeable label.See, e.g., FIG. 29. In certain embodiments depicted in FIG. 29, thehairpin structure is such that, when the first addressable portion Z1hybridizes to the second addressable portion Z2, the first addressableportion is suitable for ligation to the 3′-end of the hairpin structure.In certain embodiments, the ligation product is ligated to the 3′-end ofthe hairpin structure to form a detectable complex.

In certain embodiments, the beads comprising the codeable label arepolystyrene beads embedded with a magnetic material such as ferrite,making such beads magnetic and denser than certain buffers. In certainembodiments, the codeable labels may be photon-emitting particlesembedded or attached to the beads. In certain embodiments, thephoton-emitting particles may produce signals at unique wavelengths, andthe number of particles for each bead may be varied, allowing differentsignal intensities and wavelengths to increase the number of uniquebeads.

In certain embodiments, after a ligation reaction, the mixture is addedto a vessel containing the codeable labels, as shown in FIG. 27(A). Incertain embodiments, prior to adding the mixture to the vesselcontaining beads comprising codeable labels, after the ligationreaction, one may substantially remove the first oligonucleotide probenot in a ligation product by chemistry that decomposes probes startingat the 3′-phosphate end. In such embodiments, the ligation productshould not be decomposed because the biotin molecule is at the 3′-end.In certain embodiments, targets could be substantially removed byenzymatic digestion, such as with RNase, as a non-limiting example. Incertain embodiments, filters may be used to remove RNA or DNA targets.

In certain embodiments shown in FIG. 27(B), the detectable complexes areexposed to a streptavidin-coated magnet and the beads are attracted tothe magnet. In certain embodiments shown in FIG. 27(C), the magnet isturned off, and detectable complexes with biotin molecules remainattached to the streptavidin-coated magnet, while beads comprisingcodeable labels not in detectable complexes fall away from thestreptavidin-coated magnet. In certain embodiments, a second magnet maybe placed on the bottom of the vessel, such that the second magnetremoves unbound beads from the streptavidin-coated magnet, but does notremove the beads in detectable complexes bound to thestreptavidin-coated magnet by a biotin molecule. In certain embodimentsshown in FIGS. 27(D) and 27(E), one repeats the process of turning onthe magnet, allowing the detectable complexes to attach to thestreptavidin surface, turning off the magnet, and allowing beadscomprising codeable labels not in detectable complexes to fall off. Incertain embodiments shown in FIG. 27(F), the streptavidin-coated magnetis then removed with the bound detectable complexes. In certainembodiments, a camera may be used to evaluate codeable labels to countand identify the detectable complexes attached to the magnet. The numberof detectable complexes is then used to quantitate the target moleculesin the sample.

In certain embodiments, multiple wells may be used that each contain thesame sets of a given number of different beads with codeable labels anddifferent addressable portions. In certain embodiments, each wellincludes a set of first oligonucleotide probes that have differentaddressable portions that are complementary to the addressable portionsof the different beads. In separate wells, however, the firstoligonucleotide probes may have different target specific portionscomplementary to different targets. In certain such embodimentsdifferent multiplex assays may be performed in different wells thatemploy the same sets of different beads. In certain embodiments, if thenumber of multiplex assays per well is 1,000, then 1,000 different beadswould enable 384,000 different assays in a 384 well plate.

Quantitation of Targets

In certain embodiments of the invention, one can quantitate the amountof one or more targets, such as target nucleic acid sequences, in asample. Quantitation can be applied to any of the methods discussedabove with respect to detecting the presence or absence of targets. Forexample, and without limitation, one can quantitate the number ofdifferent particular nucleic acid sequences in a sample, including butnot limited to, the number of various alleles at one or more loci, thenumber of particular single nucleotide polymorphisms, and the number ofparticular splice variants.

In certain embodiments, to quantitate the amount of a target nucleicacid sequence in a sample, one determines the amount of a particularligation product in a sample by determining the amount of the particularcombination of codeable labels for that ligation product. In certainsuch embodiments, one may determine the quantity of a particular targetsequence in a biological sample without subjecting the biological sampleto an amplification reaction such as polymerase chain reaction.

Also, in certain embodiments that employ quantum dots as the labels, thenumber of combinations of sets of quantum dots that are determinedcorrelates directly to the actual number of ligation products in asample. Thus, in such embodiments, one need not compare the level ofintensity of a fluorescent signal to a control signal to evaluate thenumber of ligation products in the sample.

Quantitation of nucleic acid sequences may have many usefulapplications. An organism's genetic makeup is determined by the genescontained within the genome of that organism. Genes are composed of longstrands or deoxyribonucleic acid (DNA) polymers that encode theinformation needed to make proteins. Properties, capabilities, andtraits of an organism often are related to the types and amounts ofproteins that are, or are not, being produced by that organism.

A protein can be produced from a gene as follows. First, the DNA of thegene that encodes a protein, for example, protein “X”, is converted intoribonucleic acid (RNA) by a process known as “transcription.” Duringtranscription, a single-stranded complementary RNA copy of the gene ismade. Next, this RNA copy, referred to as protein X messenger RNA(mRNA), is used by the cell's biochemical machinery to make protein X, aprocess referred to as “translation.” Basically, the cell's proteinmanufacturing machinery binds to the mRNA, “reads” the RNA code, and“translates” it into the amino acid sequence of protein X. In summary,DNA is transcribed to make mRNA, which is translated to make proteins.

The amount of protein X that is produced by a cell often is largelydependent on the amount of protein X mRNA that is present within thecell. The amount of protein X mRNA within a cell is due, at least inpart, to the degree to which gene X is expressed. Whether a particulargene is expressed, and if so, to what level, may have a significantimpact on the organism.

For example, the protein insulin, among other things, regulates thelevel of blood glucose. The amount of insulin that is produced in anindividual can determine whether that individual is healthy or not.Insulin deficiency results in diabetes, a potentially fatal disease.Diabetic individuals typically have low levels of insulin mRNA and thuswill produce low levels of insulin, while healthy individuals typicallyhave higher levels of insulin mRNA and produce normal levels of insulin.

Another human disease typically due to abnormally low gene expression isTay-Sachs disease. Children with Tay-Sachs disease lack, or aredeficient in, a protein(s) required for sphingolipid breakdown. Thesechildren, therefore, have abnormally high levels of sphingolipidscausing nervous system disorders that may result in death.

It is useful to identify and detect additional genetic-baseddiseases/disorders that are caused by gene over- or under-expression.Additionally, cancer and certain other known diseases or disorders canbe detected by, or are related to, the over- or under-expression ofcertain genes. For example, men with prostate cancer typically produceabnormally high levels of prostate specific antigen (PSA); and proteinsfrom tumor suppressor genes are believed to play critical roles in thedevelopment of many types of cancer.

In certain embodiments, using nucleic acid technology, minute amounts ofa biological sample can typically provide sufficient material tosimultaneously test for many different diseases, disorders, andpredispositions. Additionally, there are numerous other situations whereit would be desirable to quantify the amount of specific target nucleicacids, e.g., mRNA, in a cell or organism, a process sometimes referredto as “gene expression profiling.” When the quantity of a particulartarget nucleic acid within, for example, a specific cell-type or tissue,or an individual is known, in certain cases one may start to compile agene expression profile for that cell-type, tissue, or individual.Comparing an individual's gene expression profile with known expressionprofiles may allow the diagnosis of certain diseases or disorders incertain cases. Predispositions or the susceptibility to developingcertain diseases or disorders in the future may also be identified byevaluating gene expression profiles in certain cases. Gene expressionprofile analysis may also be useful for, among other things, geneticcounseling and forensic testing in certain cases. In certainembodiments, gene expression profiles for one or more target nucleicacid sequences may be compiled using the quantitative informationobtained according to the inventive methods disclosed herein.

In certain embodiments, when the gene expression levels for severaltarget nucleic acid sequences for a sample are known, a gene expressionprofile for that sample can be compiled and compared with other samples.For example, but without limitation, samples may be obtained from twoaliquots of cells from the same cell population, wherein one aliquot wasgrown in the presence of a chemical compound or drug and the otheraliquot was not. By comparing the gene expression profiles for cellsgrown in the presence of drug with those grown in the absence of drug,one may be able to determine the drug effect on the expression ofparticular target genes.

Protein Detection

In certain embodiments of the invention, methods of detecting thepresence or absence of at least two target proteins in a sample areprovided. In certain embodiments, the method comprises combining thesample with a different probe set specific for each of the at least twotarget proteins, each probe set comprising (a) at least one separatingbead, comprising a magnetic particle, a first codeable label comprisingat least two labels, and a first target-specific probe, wherein thefirst codeable label is specific for the first target-specific probe,and (b) at least one detecting bead, comprising a second codeable labelcomprising at least two labels, and a second target-specific probe,wherein the second codeable label is specific for the secondtarget-specific probe. In certain such embodiments, the first and secondtarget-specific probes bind to different portions of the same targetprotein. In certain embodiments, a detectable complex is formed if thetarget protein is present in the sample. In certain embodiments, themethod further comprises detecting the presence or absence of the atleast two different proteins in the sample by counting the detectablecomplex for each of the at least two target proteins.

In certain embodiments, the target-specific probes are antibodies orfragments of antibodies.

For example, in certain embodiments, the first target-specific probe isa first antibody that can bind specifically to a first portion of aparticular target protein, and the second target-specific probe is asecond antibody that can bind specifically to a different second portionof the target protein. As a non-limiting example of preparing antibodiesfor certain such embodiments, one fragment of the target protein is usedto generate a first antibody, and a different fragment of the targetprotein is used to generate a second antibody. The first antibody isattached to a magnetic separating moiety. The second antibody isattached to a codeable label.

In certain embodiments, in the presence of the target protein, the firstantibody and second antibody specifically bind to different portions ofthe target protein, so that binding of either the first antibody or thesecond antibody to the protein does not inhibit the binding of the otherantibody to the protein. If the target protein is present, a detectablecomplex forms. In certain embodiments, the detectable complex may beseparated from unbound antibodies using the separating techniquesdiscussed above. By counting the unique combinations of codeable labels,one detects the presence of absence of particular detectable complexes,which indicates the presence or absence of the target protein in thesample. In certain embodiments, one may determine the quantity of atarget protein or proteins in a sample by determining the number ofdetectable complexes.

Certain Embodiments of Kits

In certain embodiments, kits for detecting target nucleic acid sequencesin a sample are provided. In certain embodiments, the kits comprise adifferent bead set specific for each of the target nucleic acidsequences. In certain embodiments, each different the bead set comprises(a) at least one separating bead, comprising a magnetic particle, afirst codeable label comprising two or more labels, and a firsttarget-specific probe, wherein the first codeable label is specific forthe first target-specific probe, and (b) at least one detecting bead,comprising a second codeable label comprising a set of two or morelabels, and a second target-specific probe, wherein the second codeablelabel is specific for the second target-specific probe; and wherein thefirst codeable label is detectably different from the second codeablelabel. In certain embodiments, the target-specific probes in each setare suitable for ligation together when hybridized adjacent to oneanother on a complementary target sequence.

In certain embodiments, the kit comprises a ligation agent. In certainembodiments, the ligation agent is a ligase. In certain embodiments, theligation agent is a thermostable ligase. In certain embodiments, thethermostable ligase is selected from at least one of Tth ligase, Taqligase, and Pfu ligase.

EXAMPLES

The following examples illustrate certain embodiments of the invention,and do not limit the scope of the invention in any way.

Example 1

The following experiment showed that probes that were attached to beadshybridized to a target nucleic acid sequence and were ligated in anoligonucleotide ligation assay (OLA). The amount of ligated product thatwas produced with probes attached to beads was compared to the amount ofligation product produced by the same probes that were not attached tobeads.

Magnetic beads were attached to oligonucleotide probes. Magnetic beadscoated with streptavidin were obtained from Seradyne (Sera-Mag., Lot No.113564). Biotin was attached to oligonucleotide probes (target-specificoligonucleotides (TSO probes)) as follows. Biotin-labeledoligonucleotide probes (target-specific oligonucleotide probes (TSOprobes)) were synthesized by Applied Biosystems, Inc. (Foster City,Calif.). The TSO probes were designed to hybridize to a target nucleicacid sequence. The sequence of the TSO probe is given in Table 2 below.Approximately 100 μl of the streptavidin-coated magnetic beads (1 mg/ml)were added to 100 μl of a biotin-TSO probes (10 μM) into 0.2 ml tubes.The mixture was incubated for 60 minutes at 4° C., allowing the biotinto bind to the streptavidin.

TABLE 2 TSO TAC GGA TGC TCA CTA CGC TAG GTT TTT TTT (SEQ ID 1) TTT TTTTTT T pTSO TTT TTT TTT TTT TTT TTT TTA TGC CTC GTG (SEQ ID 2) ACT GCTACC A synthetic AAA AAA AAA AAA AAA AAA CCT AGC GTA GTG target AGC ATCCGT ATG GTA GCA GTC ACG AGG CAT (SEQ ID 3) AAA AAA AAA AAA AAA AAA AA399-Taqman TTT TTT TTT TTT TTT TTT TTA TGC CTC GTG (SEQ ID 4) ACT GCTACC ATA CGG ATG CTC ACT ACG CTA GGT TTT TTT TTT TTT TTT TT

The magnetic beads were then washed to remove unbound TSO probes fromthe beads as follows. Forty μl of PBS buffer was added to the 20 μl ofthe mixture in each of the 0.2 ml tubes. The PBS buffer comprised:

KPO4 (dibasic) 1.82 g/l NaPO4 (monobasic) 0.22 g/l NaCl 8.76 g/ladjusted to pH 7.4.The sample was vortexed briefly then placed on top of a magnet for 4minutes. After 4 minutes, 40 μl was removed from each of the 0.2 mltubes while the 0.2 ml tubes were still on the magnet. The 40 μl ofsupernatant from each tube was stored in a 1 ml tube for later analysis.The magnetic separation and washing were repeated 3 more times, for atotal of 4 magnetic separations and washings.

A second oligonucleotide probe (pTSO probe) was designed to hybridize tothe target nucleic acid sequence next to the region that iscomplementary to the TSO probe sequence. The pTSO probe is adjacent tothe TSO probe when both TSO and pTSO probes hybridize to the targetnucleic acid sequence. When both TSO and pTSO probes hybridize to thetarget nucleic acid sequence they can be ligated together in a ligationreaction. Biotin-labeled pTSO probes were synthesized by AppliedBiosystems, Inc. (Foster City, Calif.). The sequence of the pTSO probeis given in Table 2.

Fluorescent beads coated with streptavidin were obtained from BangsLaboratories (Fisher, Ind.).

Ligation reactions (OLA) were performed. Eleven different reactions wereprepared by mixing the magnetic beads with the attached TSO probes, thefluorescently-labeled streptavidin-coated beads, the pTSO probes withbiotin attached, a ligase, and the synthetic target nucleic acid in adifferent concentration for each reaction. The sequence of the synthetictarget is given in Table 2. The synthetic target nucleic acid sequenceconcentrations in each of the 11 reactions, descending in orders ofmagnitude, ranged from 10 nM to as little as 10 aM (see FIG. 15 foractual concentrations). There was one control with no synthetic targetnucleic acid sequence.

The recipe for the reactions was as follows:

2 μl 10×Taq ligase buffer

2 μl biotin-labeled pTSO probe (100 nM)

2 μl Fluorescent beads (100 μg/ml)

16 μl magnetic beads attached to TSO (1 mg/ml)

0.5 μl ligase (40 U/μl)

2 μl synthetic target (10 nM-10 aM)

The ligation reactions were incubated at for ten cycles of 15 seconds at95° C., then cooled to 50° C. for 20 minutes on an ABI 9700 ThermalCycler. Taq Ligase and ligase buffer were obtained from New EnglandBiolabs (Catalog No. MO208).

After the ligation reaction, the magnetic beads were washed to removeany unligated fluorescent beads. Forty μl of PBS buffer was added to the20 μl of the mixture in each of the 0.2 ml tubes. The sample wasvortexed briefly then placed on top of a magnet for 4 minutes. After 4minutes, 40 μl was removed from each of the 0.2 ml tubes while the 0.2ml tubes were still on the magnet. The 40 μl of supernatant from eachtube was stored in a 1 ml tube for later analysis. The magneticseparation and washing were repeated 3 more times, for a total of 4magnetic separations and washings.

In addition to the 11 OLA reactions including beads, 11 other OLAreactions, were performed without beads using the same TSO and pTSOprobes without beads, the same ligase, the same ligase buffer, and thesame concentrations of the target nucleic acid sequence. The recipe forthose reactions was as follows:

2 μl 10× ligase buffer

11.5 μl water

2 μl biotin-labeled TSO probe (100 nM)

2 μl biotin-labeled pTSO probe (100 nM)

0.5 μl ligase (40 U/μl)

2 μl synthetic target (10 nM-10 aM)

Magnetic beads from each of the reactions of varying concentration werethen removed from the reactions with a magnet. The beads were washed asdescribed above and transferred to a separate detection vessel for eachdifferent reaction. The detection vessel was a Petroff-Hausser countingchamber (VWR, catalog No. 15170-048). The fluorescent beads of theligation products were then detected with an ABI 7700 (AppliedBiosystems, Foster City, Calif.).

FIG. 16 shows five photographs of bead pairs (containing fluorescentbeads) visible from five of the reactions and a photograph of magneticbeads only by visible light microscopy. One “view” in each panelrepresents approximately 0.5 μl average volume of the ligation products.FIG. 16 shows that the fluorescent beads have been successfully pairedto magnetic beads, washed, and transferred.

OLA reactions with the beads attached to probes were compared to each ofthe counterpart target concentration OLA reactions without beadsattached to probes. The ligation products were measured with a Taqman™analysis.

The 399-Taqman probe was provided by Applied Biosystems (Foster City,Calif.). The sequence of the probe is given in Table 2. The Taqman™probes and procedures for using them are described in, e.g., U.S. Pat.No. 5,538,848. Taqman™ probes work by the 5′-nuclease activity of a DNApolymerase. A Taqman™ probe hybridizes to a target nucleic acid sequenceif the target is present. The Taqman probe has a fluorescent molecule onone end of the probe, and a quenching molecule at the other end of theprobe. When the probe is intact with both the fluorescent molecule andquenching molecule attached, there is no fluorescence.

Primers are added that also hybridize to the target nucleic acidsequence. A polymerization reaction is then started at the primer, whichadds nucleotides to the end of the primer. The Taqman™ probe on thetarget nucleic acid sequence is cleaved during the polymerizationreaction as a result of the strand replacement that occurs during DNApolymerization. That cleavage frees the fluorescent molecule from thepresence of the quenching molecule on the probe, which results in thefluorescent molecule fluorescing. Thus, the detection of fluorescenceindicates the presence of the particular target nucleic acid sequenceinvolved in the polymerase reaction.

Moreover, the level of fluorescence correlates to the amount of targetnucleic acid sequence in a sample (the higher the level of fluorescence,the higher the amount of target nucleic acid sequence). A Ct value forthe level of fluorescence for a given sample can be calculated. Ctvalues are inversely related to the level of fluorescence. In otherwords, the lower the Ct value, the higher the level of fluorescence.

The recipe for the Taqman™ analysis on each of the products from the 22different OLA reactions was as follows

1 μl 399-Taqman™ probe (5 μM)

2.5 μl 116/115 primers (10 μM)

6.5 μl water

12.5 μl 2× Master Mix

2.5 μl sample from the previous OLA reaction

The Taqman™ reactions were incubated at 95° C. for ten minutes, followedby cycles comprising a first step of 15 seconds at 95° C., and a secondstep of 1 minute at 60° C. After a certain number of cycles, a signalappears. The number of cycles a reaction undergoes before a signalappears is recorded and referred to as the Ct value. The sequences ofthe 116/115 primers for the Taqman™ reactions are given in Table 2.Master Mix was obtained from Applied Biosystems (Cat. No. 4318739).

Taqman assays of the products of each of the OLA reactions with thebeads present were compared to the products of the counterpart targetnucleic acid sequence concentration OLA reactions without the beads. Theresults are shown in FIG. 15. The Taqman analysis in FIG. 15 shows thehybridization (less than one hour) and target sensitivity (greater than1 fM) for probes in solution and probes attached to beads. The detectionoccurred with a reaction time of about 1 hour.

Example 2

The following experiment was performed to determine the number of beadpairs that were detected when different amounts of synthetic targetmolecules were used.

Polystyrene beads were obtained from Bangs (No. PA05N/2057). The beadswere approximately 3.1 μm in diameter, had —NH₂ groups on the surface ata density of 10⁵ sites per μm² (according to the manufacturer), and hada density of 1.073 g/cm³.

As discussed in Example 1, the ISO probe was designed to hybridize to atarget nucleic acid sequence. The sequence of the TSO probe is given inTable 2. The TSO probe was attached at the 5′ end to the amine groups onthe Bangs beads, to create TSO-beads, according to the followingprotocol.

The beads (1.0 ml at 100 mg/ml) were washed twice in 10.0 ml of PBS aspreviously described. After the second wash, the beads were resuspendedin 10.0 ml of glutaraldehyde solution (10% glutaraldehyde in PBS). Thebeads were allowed to react at room temperature for two hours withcontinuous mixing. The beads were then washed twice in PBS as previouslydescribed, and resuspended in 5 ml of PBS. The amine-coupled TSO probewas placed in 5 ml of PBS and combined with the 5 ml solution containingthe beads. The mixture was allowed to react at room temperature for 2-4hours with continuous mixing. The beads were then washed again, andresuspended in 10 ml of PBS containing 0.1% Tween-20. The resuspendedbeads were incubated for 30 minutes. The beads were then washed oncemore, and resuspended in a storage 10 ml of PBS containing 0.1%Tween-20.

As discussed above, a second oligonucleotide probe (pTSO probe) wasdesigned to hybridize to the target nucleic acid sequence next to theregion that is complementary to the TSO probe sequence. The pTSO probeis adjacent to the TSO probe when both the ISO and pTSO probes hybridizeto the target nucleic acid sequence. When both TSO and pTSO probeshybridize to the target nucleic acid sequence they can be ligatedtogether in a ligation reaction. Biotin-labeled pTSO probes weresynthesized by, and obtained from Applied Biosystems, Inc. (Foster City,Calif.). The sequence of pTSO is given in Table 2.

Oligonucleotide Ligation Assay (OLA) reactions were performed. Eightdifferent reactions were prepared by mixing the TSO-beads with thebiotin-labeled pTSO, ligase, and synthetic target at varyingconcentrations. The reaction mixture was as follows:

2 μl 10x ligase buffer 2 μl biotin-labeled pTSO (100 nM) 2 μl TSO-beads14 μl  ddH₂O 0.25 μl   ligase (40 U/μl) 2 μl synthetic target (atvarying concentration of 1 nM down to 1 fM, plus one reaction with notarget)

The ligation reactions were incubated at for ten cycles of 15 seconds at95° C., then cooled to 50° C. for 20 minutes on an ABI 9700 ThermalCycler. Taq Ligase and ligase buffer were obtained from New EnglandBiolabs (Catalog No. MO208).

Streptavidin-coated magnetic beads were obtained from Seradyne(Sera-Mag., Lot No. 113564) which were made of polystyrene containing40% magnetite (Fe₃O₄), were approximately 1.0 μm in diameter, and had adensity of 1.5 g/cm². The streptavidin was on the surface of the beadsat a density of 10⁷ sites per bead.

In separate reactions, 20 μl of each of the eight different OLAreactions were added to 20 μl of the streptavidin-coated beads (10⁶beads/μl). These mixtures were incubated at 4° C. for 1 hour.

After incubation, each of the 40 μl OLA streptavidin-coated beadmixtures were vortexed briefly, then sonicated for 10 seconds at powerlevel 9 on a VWR “Aquasonic” ultrasonic cleaner. After sonication, 20 μlof each of the mixtures were placed into separate 0.2 ml tubes thensubjected to magnetic separation and washing.

The procedure for magnetic separation and washing was as follows. Fortyμl of PBS buffer was added to the 20 μl of the mixture in each of the0.2 ml tubes. The sample was vortexed briefly then placed on top of amagnet for 4 minutes. After 4 minutes, 40 μl was removed from each ofthe 0.2 ml tubes while the 0.2 ml tubes were still on the magnet. The 40μl of supernatant from each tube was stored in a 1 ml tube for lateranalysis. The magnetic separation and washing were repeated 3 moretimes, for a total of 4 magnetic separations and washings.

After the magnetic separation and washing, each of the supernatantscollected in the 1 ml tubes were centrifuged for 5 minutes.

A portion of the remainder of each of the eight different OLA reactionsleft in the 0.2 ml tubes after magnetic separation and washing wassubjected to Taqman™ analysis to determine the number of ligated beadpairs in each reaction. In addition, Taqman™ analysis was performed on aportion of each of the supernatants in the 1 ml tubes to determine howmany bead pairs were not separated from the wash buffer by the magneticseparation procedure.

The 399-Taqman probes were provided by Applied Biosystems (Foster City,Calif.). The sequence of the probe is given in Table 2. In general,Taqman™ probes and procedures for using them are described in, e.g.,U.S. Pat. No. 5,538,848. Taqman™ probes work by the 5′-nuclease activityof a DNA polymerase. A Taqman™ probe hybridizes to a target nucleic acidsequence if the target is present. The Taqman probe has a fluorescentmolecule on one end of the probe, and a quenching molecule at the otherend of the probe. When the probe is intact with both the fluorescentmolecule and quenching molecule attached, there is no fluorescence.

Primers are added that also hybridize to the target nucleic acidsequence. A polymerization reaction is then started at the primer, whichadds nucleotides to the end of the primer. The Taqman™ probe on thetarget nucleic acid sequence is cleaved during the polymerizationreaction as a result of the strand replacement that occurs during DNApolymerization. That cleavage frees the fluorescent molecule from thepresence of the quenching molecule on the probe, which results in thefluorescent molecule fluorescing. Thus, the detection of fluorescenceindicates the presence of the particular target nucleic acid sequenceinvolved in the polymerase reaction.

Moreover, the level of fluorescence correlates to the amount of targetnucleic acid sequence in a sample (the higher the level of fluorescence,the higher the amount of target nucleic acid sequence). A Ct value forthe level of fluorescence for a given sample can be calculated. Ctvalues are inversely related to the level of fluorescence. In otherwords, the lower the Ct value, the higher the level of fluorescence.

The recipe for the Taqman™ analysis on each of the products from the 8different OLA reactions and 8 saved supernatants was as follows:

1 μl 399-Taqman™ probe (5 μM)

2.5 μl 116/115 primers (10 μM)

6.5 μl water

12.5 μl 2× Master Mix

2.5 μl sample from the previous OLA reaction

The Taqman™ reactions were incubated at 95° C. for ten minutes, followedby cycles comprising a first step of 15 seconds at 95° C., and a secondstep of 1 minute at 60° C. The sequences of the 116/115 primers for theTaqman™ reactions are given in Table 2. Master Mix was obtained fromApplied Biosystems (Cat. No. 4318739).

In addition, portions of the bead pairs from each reaction and from eachof the supernatants collected were plated separately on grids. The gridswere visually inspected in order to calculate the total number of beadpairs in each of the supernatants and in each of the reactions.

The results of the Taqman™ analyses and visual inspections are shown inFIG. 17 and in Tables 3, 4, and 5.

Number of Bead-Pairs and Beads in Each Reaction as Determined by VisualInspection

TABLE 3 Bead % pairs Unpaired Target pairs per Total No. per beads perConc. Target No.. grid Bead pairs target grid 100 pM 1.2 × 10⁹ 191 764000.0063 1 10 pM 1.2 × 10⁸ 208 83200 0.07 0 1 pM 1.2 × 10⁷ 150 60000 0.5020 100 fM 1.2 × 10⁶ 85 34000 2.8 40 10 fM 1.2 × 10⁵ 11 4400 3.7 7 1 fM1.2 × 10⁴ 4 1600 13 5 100 aM 1.2 × 10³ 4 1600 133 3 0 0 3 1200 NA 9

Table 3 shows the total number of bead pairs counted in the grid foreach reaction, and the number of bead pairs calculated to have beenmagnetically separated in each reaction in view of the percentage of thereaction material placed on the grid. It also shows the calculatedpercentage of bead pairs generated per target molecule present in thesample, and the number of beads not incorporated into a bead pair.

Calculated Number of Successful Ligations Determined by Taqman Analysis

TABLE 4 % ligations Pre- Pre- No. ligations per target Post- Post-Target separation separation before before separation separation Conc.Ct yield (fM) separation separation Ct yield (fM) 100 pM 16.35 10157 1.2× 10⁸ 10 16.66 7917.0 10 pM 15.28 23579 2.8 × 10⁸ 236 15.9 14412.9 1 pM17.17 5328.8 6.4 × 10⁷ 533 17.28 4870.5 100 fM 20.43 407.2 4.9 × 10⁶ 40720.98 264.8 10 fM 22.65 70.6 8.5 × 10⁵ 709 25.92 5.4 1 fM 23.71 30.8 3.7× 10⁵ 3078 27.86 1.2 100 aM 23.99 24.6 3.0 × 10⁵ 24560 29.43 0.3 0 NA NA28.63 0.6

Table 4 shows the calculated yield of ligation products as determined byTaqman analysis. Table 4 shows the concentration and number of ligationproducts calculated to have been present before and after the magneticseparation procedures, and the calculated percentage of ligationsgenerated per target in the sample.

Number of Ligations After Separation by Taqman Analysis

TABLE 5 % ligations No. ligations after after Target Conc. separationseperation 100 pM 9.5 × 10⁷ 78  10 pM 1.7 × 10⁸ 61  1 pM 5.9 × 10⁷ 91100 fM 3.2 × 10⁶ 65  10 fM 6.5 × 10⁴ 8  1 fM 1.4 × 10⁴ 4 100 aM 4.1 ×10³ 1  0 7.7 × 10³ NA

Table 5 shows the number of ligation products calculated to have beenpresent in each of the reactions after the separation procedures. Table5 also shows the calculated percentage of ligations generated in each ofthe OLA reactions that were separated from the wash buffer and otherreactants by the magnetic separation procedures. The number of ligationproducts were determined by Taqman assays.

Example 3

Coded polystyrene beads of approximately 5.6 μm diameter were purchasedfrom Luminex (Luminex Corp., Austin, Tex.). The beads purchased had beenimpregnated with two colors of dye, and possess different intensities ofdye, creating unique optical code detectable by a Luminex 100 FlowCytometer. Oligonucleotides were attached to the surface of the beadsusing the NH₂ ester chemistry described in the Luminex manuals. One polyethylene glycol (PEG) linker was included between the amine group on theLuminex bead and 5′-end of the attached oligonucleotide. Theoligonucleotide comprised a 20 base primer sequence and a 20 base longsequence (referred to as an addressable portion) that was designed forspecific hybridization with minimal cross reactivity at a specifictemperature. The primer sequence was located between the PEG linker andthe addressable portion. After the oligonucleotides were attached, thebeads were washed four times with a phosphate buffered saline PBS, asdescribed in Example 1 above, with 0.1% Tween-20 at pH 7.4, in order toremove oligonucleotides not covalently attached to beads. The washingwas between 50-65° C., and lasted 20 minutes. Sequences of the twodifferent oligonucleotides with two different addressable portions thatwere attached to the two differently coded beads are designated Z1 andZ2 as shown in Table 6 below. The addressable portions of the sequencesof Z1 and Z2 are shown in bold.

TABLE 6 Z1 NH₂-PEG-GCTGATGCTACTGGATCGCT (SEQ ID 5) ACCGTGACCCTTCCGA Z2NH₂-PEG-GCTTGCCTGCTCGACTTAGA (SEQ ID 6) AATCGGTCTCGTCCTTCA ASO1p-GCGTAATCGTTGCTTCATAG CCTGGCAGTAAATTCTA (SEQ ID 7) G ASO2p-ACAGGAGTGAGTCTTTAGG CCTGGCAGTAAATTCTA (SEQ ID 8) C Z3CTATGAAGCAACGATTACGC TCGGAAGGGTCACGGT (SEQ ID 9) Z4 CCTAAAGACTCACTGCTGTTGAAGGACGAGACCGATT (SEQ ID 10) LSO p-CTCAACCTTACTTGAGGC (SEQ ID 11)TGGTAGCAGTCACGAGGCAT-PEG-BIO

A set of first Luminex beads (B1) and a set of second Luminex beads (B2)(10,000 beads per set) were used. The first bead (B1) comprised a firstcodeable label and oligonucleotide Z1. The second bead (B2) comprised asecond codeable label and oligonucleotide Z2.

Ligation probes were used as follows. Two probes were allele specificoligos (ASO1 and ASO2, shown in Table 6) which comprised a targetspecific portion complementary to the target sequence containing aparticular single nucleotide polymorphorism (SNP). The ASO's weredesigned with the base complementary to the SNP located at the 3′ end ofthe probe. Thus, ASO1 and ASO2 had different bases at their 3′-ends.

ASO1 comprised an addressable portion 5′ to the target specific portion.ASO2 comprised a different addressable portion 5′ to the target specificportion. Both ASO's were 40 bases in length. The twenty bases at the3′-ends of the ASO's were target specific portions, while the 20 basesat the 5′-ends were addressable portions. The addressable portions ofASO1 and ASO2 are shown in bold in Table 6.

A locus specific oligo (LSO) was also provided. The sequence of LSO isshown in Table 6. LSO comprised a target specific portion complementaryto the target adjacent to the part of the target that was complementaryto the target specific portions of the ASOs, such that LSO and ASO1 orASO2, when hybridized to the target, were suitable for ligationtogether. LSO had a biotin molecule attached to the 3′ end with a PEGlinker. The PEG linker was disposed between the last 3′ nucleotide andthe biotin molecule. LSO was 40 bases in length. Twenty bases at the5′-end of the LSO were the target specific portion, while the 20 basesat the 3′-end of LSO were complementary to a particular Taqman primer.

A synthetic template Z3 comprised a 3′-end sequence complementary to theaddressable portion of oligonucleotide Z1 on bead B1, and comprised a5′-end sequence complementary to the addressable portion on ASO1, suchthat ASO1 and oligonucleotide Z1 are suitable for ligation whenhybridized to synthetic template Z3.

A synthetic template Z4 comprised a 3′-end sequence complementary to theaddressable portion of oligonucleotide Z2 on bead B2, and comprised a5′-end sequence complementary to the addressable portion on ASO2, suchthat ASO2 and oligonucleotide Z2 are suitable for ligation whenhybridized to synthetic template Z4.

The target was genomic DNA heated for 30 minutes at 100° C. The targetwas known (verified by other means) to contain a single nucleotidepolymorphism (SNP) complementary to ASO1 but not complementary to ASO2.

The ligation reaction volume was 6.75 microliters. The reaction mixturewas as follows.

0.5 μl 10x Ligase Buffer 0.5 μl ASO1 probes at 100 nM (3 × 10¹⁰ probes)0.5 μl ASO2 probes at 100 nM (3 × 10¹⁰ probes) 0.5 μl LSO probes at 100nM (3 × 10¹⁰ probes) 0.5 μl Template Z3 at 10 nM concentration (3 × 10⁹molecules) 0.5 μl Template Z4 at 10 nM concentration (3 × 10⁹ molecules)  1 μl solution of B1 (10,000 beads)   1 μl solution of B2 (10,000beads) 0.25 μl  Taq Ligase (40 units/μl) 0.5 μl heated gDNA (100 ng/μl)

Taq Ligase and 10× ligase buffer were purchased from New England Biolabs(Catalog No. M0208). Beads were sonicated for 10 seconds before beingadded to the reaction mixture. The reaction mixture was temperaturecycled, starting at 50° C. for 5 minutes to hybridize and ligate the ASOand LSO probes on the target, and to ligate the Z1 or Z2oligonucleotides to the ASO probes on the synthetic templates. Then, thetemperature was increased to 85° C. for 15 seconds, in order to denaturethe probes from the target, and to denature the synthetic templates fromthe probes and oligonucleotides. This cycle was repeated 100 times. Atthe completion of the temperature cycles, the beads were washed 3 timesin PBS buffer 0.1% Tween-20 at 85° C.

Approximately 10 million, 1 μm diameter streptavidin-coated magneticbeads were mixed with the reaction mixture after the ligation reaction.The beads were purchased from Seradyn Inc., and were made of polystyrenewith 40% magnetite (Fe₃O₄), had a density of 1.5 g/cm³, and had astreptavidin coating (10⁶-10⁶ streptavidin molecules per bead). Prior touse, the magnetic beads were washed 5 times with PBS buffer 0.1%Tween-20 and 0.1% BSA and sonicated for 10 seconds. The binding bufferused to bind the magnetic beads to biotin molecules was PBS with 0.1%Tween-20 in a volume of 20 μl (10 μl reaction mixture from the ligationreaction, 10 μl magnetic beads). The 20 μl volume of magnetic beads andreaction mixture from the ligation reaction formed the binding mixture,and was incubated in a tube for two hours at room temperature (25° C.)with continuous rotation of the tube. If the streptavidin on a magneticbead bound to the biotin on an LSO, a bead pair was formed.

After the binding mixture was incubated, the binding mixture wassonicated for 10 seconds. The binding mixture was pipetted into a firsttube that was sitting in a second tube filled with a high-densitysolution (˜1.3 g/cm³) of 6×SSC (90 mM Na Citrate, 0.9 M NaCl, pH 7.0)and 0.1% Bovine Serum Albumin (New England Biolabs). The first tube hadno bottom such that it was partially filled with the high-densitysolution. The lower-density (˜1.05 g/cm³) binding buffer of the bindingmixture remained on top of the higher density fluid without significantmixing. A magnet located below the tubes attracted the magnetic beads tothe bottom of the second tube.

Luminex beads that were not paired to magnetic beads remained toward thetop of the first tube and were disposed of by removing the first tubefrom the second tube. The removal was performed by covering the top ofthe first tube, then lifting the first tube out of the second tube.Luminex beads in detectable complexes that had been pulled down by themagnetic beads were then released from the magnet, and vortexed into ahomogenous solution. The homogeneous solution was then aspirated into aflow cytometer. The Luminex beads were read one at a time, and theiridentity determined by the unique codes on the beads (B1 or B2). Almostall the Luminex beads were B1 beads, indicating the presence of the SNPcorresponding to ASO1 (data not shown).

1. A method for quantitating a target comprising; forming a reactionmixture comprising: a sample possibly containing the target; a codeablelabel; one or more target-specific probes, wherein each target-specificprobe binds specifically to the target under selective bindingconditions; and a separating moiety; treating the reaction mixture underreaction conditions such that a detectable complex is produced when thetarget is present, and wherein the detectable complex comprises thecodeable label, the target-specific probe, and the separating moiety;and quantitating the target by counting the number of codeable labels.2. The method of claim 1, further comprising separating the detectablecomplex from codeable labels that are not included in the detectablecomplex after treating the reaction mixture and before quantitating thetarget.
 3. A method for quantitating at least two different particulartargets comprising; forming a reaction mixture comprising: a samplepossibly containing two or more different particular targets; adifferent codeable label specific for each different particular target;one or more different target-specific probes specific for each differentparticular target that bind specifically to the target under selectivebinding conditions; and a separating moiety; treating the reactionmixture under reaction conditions such that when a particular target ispresent, a detectable complex is produced, which comprises the codeablelabel specific for the particular target, the target-specific probespecific for the particular target, and the separating moiety; andquantitating each of the different particular targets by counting thenumber of codeable labels specific for each of the different particulartargets.
 4. The method of claim 3, further comprising separating anydetectable complexes produced from codeable labels that are not includedin the detectable complex after treating the reaction mixture and beforequantitating each of the different particular targets.
 5. A method forquantitating at least two different target nucleic acid sequences in asample comprising: forming a ligation reaction mixture by combining thesample with a different probe set specific for each of the at least twodifferent target nucleic acid sequences, each probe set comprising (a)at least one separating bead, comprising a magnetic particle and a firsttarget-specific probe, and (b) at least one detecting bead, comprising acodeable label, and a second target-specific probe; wherein thetarget-specific probes in each set are suitable for ligation togetherwhen hybridized adjacent to one another on a complementary targetsequence; subjecting the ligation reaction mixture to a ligationreaction, wherein adjacently hybridizing complementary target-specificprobes are ligated to one another to form a ligation product comprisingthe separating bead and the detecting bead; and quantitating each of theat least two different target nucleic acid sequences by counting thenumber of codeable labels for each different target nucleic acidsequence.
 6. The method of claim 5, separating any ligation product fromunligated separating beads and detecting beads after treating thereaction mixture and before quantitating each of the at least twodifferent target nucleic acid sequences.
 7. The method of claim 6,wherein separating the ligation product from unligated detecting andseparating beads comprises: separating the ligation product from thetarget nucleic acid sequences, and separating the ligation product fromthe sample.
 8. A method for detecting at least two different targetnucleic acid sequences in a sample comprising: forming a ligationreaction mixture by combining the sample with a different bead setspecific for each of the at least two different target nucleic acidsequences, each bead set comprising (a) at least one separating bead,comprising a magnetic particle, a codeable label comprising at least twolabels, and a first target-specific probe, wherein the first codeablelabel is specific for the first target-specific probe, and (b) at leastone detecting bead, comprising a second codeable label comprising atleast two labels and a second target-specific probe, wherein the secondcodeable label is specific for the second target-specific probe; whereinthe first codeable label is detectably different from the secondcodeable label; wherein the target-specific probes in each set aresuitable for ligation together when hybridized adjacent to one anotheron a complementary target sequence; subjecting the ligation reactionmixture to a ligation reaction, wherein adjacently hybridizingcomplementary target-specific probes are ligated to one another to forma detectable complex comprising the separating bead and the detectingbead; and quantitating the at least two different target nucleic acidsequences in the sample by quantitating the detectable complex.
 9. Themethod of any of claims 6-8, wherein the separating of the ligationproduct from the target nucleic acid sequences comprises thermaldenaturation.
 10. The method of claim 9, further comprising removing anyseparating beads that are not in a ligation product prior to thequantitating the target nucleic acid sequences.
 11. The method of claim10, wherein the removing of any separating beads that are not in aligation product comprises: placing any separating beads and ligationproducts in a density gradient, wherein the separating beads andligation products differ in density; and removing any separating beadsthat are not in a ligation product.
 12. The method of any of claims5-11, wherein the codeable label has a level of intensity that isspecific for the second target-specific probe.
 13. The method of claim12, wherein the separating bead further comprises a second codeablelabel, and wherein the second codeable label has a level of intensitythat is specific for the first target-specific probe.
 14. The method ofclaim 13, wherein each of the at least two probe sets that are specificfor target nucleic acid sequences comprise codeable labels that have thesame emission spectrum.
 15. The method of any of claims 1-14, whereinthe codeable label is one or more quantum dots.
 16. The method of any ofclaim 5-14, wherein the codeable label is one or more quantum dots andwherein the detecting bead of each probe set comprises at least 1,000quantum dots, wherein the quantum dots have predetermined wavelengthsthat make the detecting bead distinguishable from different detectingbeads.
 17. The method of claim 16, wherein the separating bead furthercomprises at least 1,000 quantum dots, wherein the quantum dots havepredetermined wavelengths that make the separating bead distinguishablefrom different separating beads.
 18. The method of any of claims 5-14,16, and 17, wherein the quantitating the at least two target nucleicacid sequences in the sample is performed in a detecting vesselcomprising a groove on one surface of the detecting vessel near amagnetic source, wherein the separating bead fits in the groove, thedetecting bead does not fit in the groove, and the ligation productsattracted to the magnetic source are aligned.
 19. The method of any ofclaims 5-14 and 16-18, wherein the ligation reaction mixture furthercomprises a ligation agent.
 20. The method of claim 19, wherein theligation agent is a ligase.
 21. The method of claim 19, wherein theligation agent is a thermostable ligase.
 22. The method of claim 21,wherein the thermostable ligase is selected from at least one of Tthligase, Taq ligase, and Pfu ligase.
 23. The method of any of claims 5-14and 16-22, wherein each separating bead differs in density from eachdetecting bead, such that the distance between any separating beads thatare not in a ligation product and the ligation product allows attractionof the ligation product to a magnetic device and does not allowattraction of the separating beads that are not in a ligation product tothe magnetic device.
 24. The method of claim 23, wherein thequantitating the ligation product occurs in the presence of the sample.25. The method of any of claims 1-24, wherein the codeable labelscomprise at least two phosphors.
 26. The method of any of claim 1-24,wherein the codeable labels comprise at least two fluorescent molecules.27. The method of claim 8, further comprising separating the detectablecomplex from unligated detecting and separating beads after the ligationreaction and prior to the quantitating the at least two different targetnucleic acid sequences.
 28. The method of claim 27, wherein separatingthe detectable complex from unligated detecting and separating beadscomprises: separating the detectable complex from the at least twodifferent target nucleic acid sequences, and separating the detectablecomplex from the sample.
 29. The method of claim 28, wherein theseparating of the detectable complex from the at least two differenttarget nucleic acid sequences comprises thermal denaturation.
 30. Themethod of claim 29, further comprising removing any separating beadsthat are not in a detectable complex prior to the quantitating the atleast two different target nucleic acid sequences.
 31. The method ofclaim 30, wherein the removing of any separating beads that are not in adetectable complex comprises: placing any separating beads anddetectable complexes in a density gradient, wherein the separating beadsand detectable complexes differ in density; and removing any separatingbeads that are not in a detectable complex.
 32. The method of any ofclaims 5-14 and 16-31, wherein the detecting bead further comprises amagnetic particle.
 33. The method of claim 32, wherein the quantitatingthe at least two target nucleic acid sequences in the sample isperformed in a detection vessel comprising a groove on one surface ofthe detection vessel near a magnetic source, wherein the groovecomprises a first end and a second end, and wherein the first codeablelabel and the second codeable label of the detectable complex arealigned within the groove with the magnetic source, such that theseparating bead of the detectable complex aligns closer to the first endthan the detecting bead of the detectable complex.
 34. A kit fordetecting target nucleic acid sequences in a sample comprising: adifferent bead set specific for each of the target nucleic acidsequences, the bead set comprising (a) at least one separating bead,comprising a magnetic particle, a first codeable label comprising two ormore labels, and a first target-specific probe, wherein the firstcodeable label is specific for the first target-specific probe, and (b)at least one detecting bead, comprising a second codeable labelcomprising a set of two or more labels, and a second target-specificprobe, wherein the second codeable label is specific for the secondtarget-specific probe; wherein the first codeable label is detectablydifferent from the second codeable label; and wherein thetarget-specific probes in each set are suitable for ligation togetherwhen hybridized adjacent to one another on a complementary targetsequence.
 35. The kit of claim 34, further comprising a ligation agent.36. The kit of claim 35, wherein the ligation agent is a ligase.
 37. Thekit of claim 35, wherein the ligation agent is a thermostable ligase.38. The kit of claim 37, wherein the thermostable ligase is selectedfrom at least one of Tth ligase, Taq ligase, and Pfu ligase.